Unlabeled Nanoparticles Research Articles

Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy.

We present a method that integrates the standard imaging tools for locating and detecting unlabeled nanoparticles (NPs) with computational tools for partitioning cell volumes and NPs counting within specified regions to evaluate their internal traffic. The method uses enhanced dark field CytoViva optical system and combines 3D reconstructions of double fluorescently labeled cells with hyperspectral images. The method allows the partitioning of each cell image into four regions: nucleus, cytoplasm, and two neighboring shells, as well as investigations across thin layers adjacent to the plasma membrane. MATLAB scripts were developed to process the images and to localize NPs in each region. Specific parameters were computed to assess the uptake efficiency: regional densities of NPs, flow densities, relative accumulation indices, and uptake ratios. The results of the method are in line with biochemical analyses. It was shown that a sort of saturation limit for intracellular NPs density is reached at high extracellular NPs concentrations. Higher NPs densities were found in the proximity of the plasma membranes. A decrease of the cell viability with increasing extracellular NPs concentration was observed and explained the negative correlation of the cell eccentricity with NPs number.

A concise detection strategy of Staphylococcus aureus using N-Succinyl-Chitosan-dopped bacteria-imprinted composite film and AIE fluorescence sensor

Staphylococcus aureus is one of the major foodborne pathogens. Efficient detection and isolation of Staphylococcus aureus from complex samples are crucial. Herein, we report a concise strategy to detect of Staphylococcus aureus with high sensitivity and specificity, based on N-Succinyl-Chitosan doping bacteria-imprinted composite film and aggregation-induced emission (AIE)-featuring fluorescence sensor. The good shaping and mechanical properties of polydimethylsiloxane provide a specific recognition site suitable for Staphylococcus aureus. For the first time, chitosan derivatives is combined with polydimethylsiloxane to prepare a two-component composite film, which possesses a remarkable absorption performance of Staphylococcus aureus using the natural excellent absorption property of chitosan. The positive charged AIE-featuring Au(I)-disulfide nanoparticles realized the quantitative characterization of Staphylococcus aureus without cooperation with bio-recognition elements. To conclude, this study provides new possibilities for the manufacture of highly efficient bacterial separators with superior performance and facilitates the application of unlabeled nanoparticles in quantitative analysis.

Intracellular Detection and Localization of Nanoparticles by Refractive Index Measurement

The measuring of nanoparticle toxicity faces an important limitation since it is based on metrics exposure, the concentration at which cells are exposed instead the true concentration inside the cells. In vitro studies of nanomaterials would benefit from the direct measuring of the true intracellular dose of nanoparticles. The objective of the present study was to state whether the intracellular detection of nanodiamonds is possible by measuring the refractive index. Based on optical diffraction tomography of treated live cells, the results show that unlabeled nanoparticles can be detected and localized inside cells. The results were confirmed by fluorescence measurements. Optical diffraction tomography paves the way to measuring the true intracellular concentrations and the localization of nanoparticles which will improve the dose-response paradigm of pharmacology and toxicology in the field of nanomaterials.

Tracking Interfacial Dynamics of a Single Nanoparticle Using Plasmonic Scattering Interferometry.

The ability to track interfacial dynamics of a single nanoparticle at the solution-solid interface is crucial for understanding physical, chemical, and biological processes, but it remains a challenge. Here, we demonstrated a plasmonic imaging technique that can track unlabeled nanoparticles at the solution-solid interface with high spatial and temporal resolutions. This technique is based on particle-induced interferometric scattering of a surface plasmonic wave, which results in a high vertical sensitivity. Using this ability, we tracked the trajectories of a single nanoparticle interacting with a surface, measured the hydrodynamically hindered diffusion of nanoparticles, and revealed the surface chemistry-dependent behavior of nanoparticles at the interface. The application for tracking formation of membranes from a lipid vesicle was demonstrated, indicating the potential for investigating a broad range of nano-objects at interfaces in a complex environment.

On-Chip Spiral Waveguides for Ultrasensitive and Rapid Detection of Nanoscale Objects.

Ultrasensitive and rapid detection of nano-objects is crucial in both fundamental studies and practical applications. Optical sensors using evanescent fields in microcavities, plasmonic resonators, and nanofibers allow label-free detection down to single molecules, but practical applications are severely hindered by long response time and device reproducibility. Here, an on-chip dense waveguide sensor to monitor single unlabeled nanoparticles in a strong optical evanescent field is demonstrated. The spiral nanowaveguide design enables two orders of magnitude enhancement in sensing area compared to a straight waveguide, significantly improving the particle capture ability and shortening the target analysis time. In addition, the measurement noise is suppressed to a level of 10-4 in the transmitted power, pushing the detection limit of single particles down to the size of 100 nm. The waveguide sensor on the silicon-on-isolator platform can be fabricated reproducibly by the conventional semiconductor processing and compatible with surface functionalization chemistries and microfluidics, which could lead to widespread use for sensing in environmental monitoring and human health.

Preparation and characterization of three-layer, isotopically labelled core-shell nanoparticles; a tool for understanding mechanisms of bioavailability

This paper reports the synthesis of three-layered, isotopically labelled 109Ag@Au@107Ag nanoparticles (NPs). It is proposed that this tool will be better able to fully quantify the relative importance of NP and ion (from NP dissolution) biouptake in future studies. A three-step hydrothermal synthesis procedure was developed. The final product had a middle Au barrier layer which was sufficiently thick to prevent the dissolution of the mono-isotopic (109Ag) core, despite the possibility of some alloying at the boundaries between Ag and Au. In addition, the 107Ag shell needed to be sufficiently thick to prevent complete dissolution over a relevant biological exposure period to prevent effects of the alloy on the bioaccumulation. In order to be fit for purpose, there must be good control of NP properties such as size and capping agent. After synthesis, a multi-method approach was used including scanning transmission electron microscopy (STEM), dynamic light scattering (DLS), uv–vis spectroscopy and centrifugal ultracentrifugation and inductively coupled plasma mass spectroscopy (ICP–MS) to quantify particle properties, including dissolution. The DLS, STEM and uv–vis data are strongly suggestive that a NP with three separate layers are produced, although it is acknowledged that some alloying cannot be completely ruled out. Dissolution data of the labelled NPs confirms that there is no dissolution of the Ag109 core NP or Au inner layer but partial dissolution of the Ag107 outer layer. In comparison, the dissolution data from the unlabeled NPs can only confirm the absence of dissolved Au. The middle barrier layer of Au helps prevent any hypothetical alloying affecting the particle properties as a tool to quantify dissolution impacts on bioavailability.

Stable non-covalent labeling of layered silicate nanoparticles for biological imaging

Layered silicate nanoparticles (LSN) are widely used in industrial applications and consumer products. They also have potential benefits in biomedical applications such as implantable devices and for drug delivery. To study how nanomaterials interact with cells and tissues, techniques to track and quantify their movement through different biological compartments are essential. While radiolabels can be very sensitive, particularly for in vivo studies, fluorescent labeling has been preferred in recent years because of the array of methods available to image and quantify fluorescent nanoparticles. However, labeling can be problematic, especially if it alters the physical properties of the nanomaterial. Herein is described a novel non-covalent labeling technique for LSN using readily available fluorescent dimeric cyanine dyes without the need to use excess amounts of dye to achieve labeling, or the need for removal of unbound dye. The approach utilizes the cationic binding properties of layered silicate clays and the multiple quaternary nitrogens associated with the dyes. Preparation of YOYO-1 labeled LSN with optimal dispersion in aqueous media is presented. The utilization of the labeled particles is then demonstrated in cell binding and uptake studies using flow cytometry and confocal microscopy. The labeled LSN are highly fluorescent, stable and exhibit identical physical properties with respect to the unlabeled nanoparticles. The general approach described here is applicable to other cyanine dyes and may be utilized more widely for labeling nanoparticles that comprise a crystalline plate structure with a high binding capacity.

Long-term prevalence of NIRF-labeled magnetic nanoparticles for the diagnostic and intraoperative imaging of inflammation

Inflammation is a very common disease worldwide. In severe cases, surgery is often the method of choice. Today, there is a general need for the implementation of image-based guidance methodologies for reliable target resection. We investigated new near infrared fluorescence (NIRF)-nanoparticles (NPs) as a simple but effective bimodal magnetic resonance imaging (MRI) and optical contrast agent for diagnosis and intraoperative imaging of inflammation. Physicochemical analysis revealed that these NPs were highly fluorescent with similar characteristics like unlabeled NPs (hydrodynamic diameter about 130 nm and zeta potential about −10 mV). NP-uptake and NIR-dye labeling was biocompatible to macrophages (no impact on cellular ATP and reactive oxygen species production). These cells could successfully be tracked with MRI and NIRF-optical imaging. I.v. injection of fluorescent NPs into mice led to highly specific T2-weighted signal of edema due to uptake by phagocytic cells and subsequent migration to the site of inflammation. NIRF signals of the edema region were well detectable for up to 4 weeks, underlining the potential of the NPs for systematic planning and flexible time scheduling in intraoperative applications. NPs were degraded over a time period of 12 weeks, which was not altered due to inflammation. Redistribution of iron might be primarily due to inflammation and not to the presence of NPs per se in a concentration suitable for imaging. Our findings highlight the potential of the NPs to be used as a suitable tool for pre- and intraoperative imaging of inflammation.

Interferometry and Fluorescence Detection for Simultaneous Analysis of Labeled and Unlabeled Nanoparticles in Solution

A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K(d) from a single binding measurement.

Targeted Delivery of Polyoxometalate Nanocomposites

Polyoxometalate/carboxymethyl chitosan nanocomposites with an average diameter of 130 nm are synthesized and labeled with fluorescein isothiocyanate (FITC) for a combined drug-carrier and cellular-monitoring approach. [Eu(β(2) -SiW(11) O(39) )(2) ](13-) /CMC nanospheres as a representative example do not display cytotoxicity for POM concentrations up to 2 mg mL(-1) . Cellular uptake of fluoresecently labelled {EuSiW(11) O(39) }/FITC-CMC nanoparticles is monitored with confocal laser scanning microscopy. Nanoparticle uptake occurs after incubation times of around 1 h and no cyctotoxic effects are observed upon prolonged treatment. The preferential location of the POM/CMC nanocomposites in the perinuclear region is furthermore verified with transmission electron microscopy investigations on unlabeled nanoparticles. Therefore, this approach is a promising dual strategy for the safe cellular transfer and monitoring of bioactive POMs.

Application of Laser Postionization Secondary Neutral Mass Spectrometry/Time-of-Flight Secondary Ion Mass Spectrometry in Nanotoxicology: Visualization of Nanosilver in Human Macrophages and Cellular Responses

Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.

Development of an Ultrasensitive Dual-Channel Flow Cytometer for the Individual Analysis of Nanosized Particles and Biomolecules

A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO(2) nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R(2) > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was approximately 90% on average for particle concentrations ranging from 1.62 x 10(5) to 3.93 x 10(7) particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.