Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles.
ABSTRACT: Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10?000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.
Project description:X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7?nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Project description:Most often the determination of the concentration of virus particles is rendered difficult by the availability of proper standards. We have adapted a static light scattering based method for the quantification of virus particles (shown for poliovirus) without the need of virus particle standards. Instead, as standards, well-characterized polymeric nanoparticle solutions are used. The method is applicable for virus particles acting as Rayleigh scatterers, i.e., virus particles with equivalent diameters up to ca. 1/10th of the wavelength of the scattered monochromatic light (?70 nm diameter). Further limitations may arise if the refractive index of the virus is unavailable or cannot be calculated based on its composition, such as in case of enveloped viruses. The method is especially relevant for preparation of virus particle concentration standards and to vaccine formulations based on attenuated or inactivated virus particles where the classical plaque forming assays cannot be applied. The method consists of: •Measuring the intensity of the light scattered by viruses suspended in an aqueous solution.•Measuring the intensity of the light scattered by polymeric nanoparticles of known concentration and comparable size with the investigated virus particle.•The concentration of virus nanoparticles can be calculated based on the two measured scattered light intensities by knowing the refractive index of the dispersing solution, of the polymer and virus nanoparticles as well as their relative sphere equivalent diameters.
Project description:Particle size distribution and stability are key attributes for the evaluation of the safety and efficacy profile of medical nanoparticles (Med-NPs). Measuring particle average size and particle size distribution is a challenging task which requires the combination of orthogonal high-resolution sizing techniques, especially in complex biological media. Unfortunately, despite its limitations, due to its accessibility, low cost, and easy handling, batch mode dynamic light scattering (DLS) is still very often used as the only approach to measure particle size distribution in the nanomedicine field. In this work the use of asymmetric flow field flow fractionation coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) was evaluated as an alternative to batch mode DLS to measure the physical properties of lipid-based nanoparticles. A robust standard operating procedure (SOPs) developed by the Nanomedicine Characterization Laboratory (EUNCL) was presented and tested to assess size stability, batch to batch consistency, and the behavior of the lipid-based nanoparticles in plasma. Orthogonal sizing techniques, such as transmission electron microscopy (TEM) and particle tracking analysis (PTA) measurements, were performed to support the results. While batch mode DLS could be applied as a fast and simple method to provide a preliminary insight into the integrity and polydispersity of samples, it was unsuitable to resolve small modifications of the particle size distribution. The introduction of nanoparticle sorting by field-flow fractionation coupled to online DLS and MALS allowed assessment of batch to batch variability and changes in the size of the lipid nanoparticles induced by the interaction with serum proteins, which are critical for quality control and regulatory aspects. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles.
Project description:To realize automatic measurement of the concentration of dust particles in the air, a theory for dust concentration measurement was developed, and a system was designed to implement the dust concentration measurement method based on laser scattering. In the study, the principle of dust concentration detection using laser scattering is studied, and the detection basis of Mie scattering theory is determined. Through simulation, the influence of the incident laser wavelength, dust particle diameter, and refractive index of dust particles on the scattered light intensity distribution are obtained for determining the scattered light intensity curves of single suspended dust particles under different characteristic parameters. A genetic algorithm was used to study the inverse particle size distribution, and the reliability of the measurement system design is proven theoretically. The dust concentration detection system, which includes a laser system, computer circuitry, air flow system, and control system, was then implemented according to the parameters obtained from the theoretical analysis. The performance of the designed system was evaluated. Experimental results show that the system performance was stable and reliable, resulting in high-precision automatic dust concentration measurement with strong anti-interference ability.
Project description:The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles.
Project description:The analysis of natural and otherwise complex samples is challenging and yields uncertainty about the accuracy and precision of measurements. Here we present a practical tool to assess relative accuracy among separation protocols for techniques using light scattering detection. Due to the highly non-linear relationship between particle size and the intensity of scattered light, a few large particles may obfuscate greater numbers of small particles. Therefore, insufficiently separated mixtures may result in an overestimate of the average measured particle size. Complete separation of complex samples is needed to mitigate this challenge. A separation protocol can be considered improved if the average measured size is smaller than a previous separation protocol. Further, the protocol resulting in the smallest average measured particle size yields the best separation among those explored. If the differential in average measured size between protocols is less than the measurement uncertainty, then the selected protocols are of equivalent precision. As a demonstration, this assessment metric is applied to optimization of cross flow (V(x)) protocols in asymmetric flow field flow fractionation (AF(4)) separation interfaced with online quasi-elastic light scattering (QELS) detection using mixtures of polystyrene beads spanning a large size range. Using this assessment metric, the V(x) parameter was modulated to improve separation until the average measured size of the mixture was in statistical agreement with the calculated average size of particles in the mixture. While we demonstrate this metric by improving AF(4) V(x) protocols, it can be applied to any given separation parameters for separation techniques that employ dynamic light scattering detectors.
Project description:Light absorption and scattering by metal nanoparticles can drive catalytic reactions at their surface via the generation of hot charge carriers, elevated temperatures, and focused electromagnetic fields. These photoinduced processes can substantially alter the shape, surface structure, and oxidation state of surface atoms of the nanoparticles and therefore significantly modify their catalytic properties. Information on such local structural and chemical change in plasmonic nanoparticles is however blurred in ensemble experiments, due to the typical large heterogeneity in sample size and shape distributions. Here, we use single-particle dark-field and Raman scattering spectroscopy to elucidate the reshaping and surface restructuring of individual silver nanodisks under plasmon excitation and during photocatalytic CO<sub>2</sub> hydrogenation. We show that silver nanoparticles reshape significantly in inert N<sub>2</sub> atmosphere, due to photothermal effects. Furthermore, by collecting the inelastic scattering during laser irradiation in a reducing gas environment, we observe intermittent light emission from silver clusters transiently formed at the nanoparticle surface. These clusters are likely to modify the photocatalytic activity of silver nanodisks and to enable detection of reaction products by enhancing their Raman signal. Our results highlight the dynamic nature of the catalytic surface of plasmonic silver nanoparticles and demonstrate the power of single-particle spectroscopic techniques to unveil their structure-activity relationship both <i>in situ</i> and in real time.
Project description:Nanoparticles have been incorporated into a range of consumer spray products, providing the potential for inadvertent inhalation by users and bystanders. The levels and characteristics of nanoparticle inhalation exposures arising from the use of such products are important inputs to risk assessments and informing dose regimes for <i>in vitro</i> and <i>in vivo</i> studies investigating hazard potentials. To date, only a small number of studies have been undertaken to explore both the aerosols generated from such products and the metal nanoparticles within them. The objective of the current study was to add to the limited data in this field by investigating a range of nano-containing spray products available within the UK. Six products were selected and the nanoparticles characterised using a combination of techniques, including: inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX) and single particle ICP-MS (spICP-MS). The aerosol produced by these products, when sprayed within a glovebox, was characterised by scanning mobility particle sizer (SMPS) and an aerodynamic particle sizer (APS). A cascade impactor with thirteen stages (NanoMOUDI) was used with one product to generate information on the size specific nanoparticle elemental distribution within the aerosol. The results demonstrated the presence of solid nanoparticles (silver, gold or silica) in each of the products at low concentrations (<13 ppm). TEM and (sp)ICP-MS provided reliable information on nanoparticle size, shape, number and mass, while the light scattering methods were less effective due to the complex matrices of the products and their lack of chemical specificity. The aerosols varied significantly across products, with particle and mass concentrations spanning 5 orders of magnitude (10 - 10<sup>6</sup> cm<sup>-3</sup> and 0.3-7600 ?g m<sup>-3</sup>, respectively). The NanoMOUDI results clearly indicated non-uniform distribution of silver within different aerosol particle size ranges.
Project description:The behavior of nanoparticles in biological systems is determined by their dimensions, size distribution, shape, surface chemistry, density, drug loading and stability; the characterization of these parameters in realistic conditions and the possibility to follow their evolution in vitro and in vivo are, in most of the cases, far from the capabilities of the standard characterization technologies. Optical techniques such as dynamic light scattering (DLS) are, in principle, well suited for in line characterization of nanoparticle, however their fail in characterizing the evolution of nanoparticle in solution where change in particle dimension and density is present. Here we present an in-line optical technique based on single particle extinction and scattering (SPES) overcoming the limitations typical of DLS and allowing for the efficient characterization of nanoparticle polydispersity, index of refraction and degradation dynamics in solution. Using SPES, we characterized the evolution of PLGA nanoparticles with different structures and drug payloads in solution and we compared the results with DLS. Our results suggest that SPES could be used as a process analytical technology for pharmaceutical nanoparticle production.
Project description:The interaction of light with metal nanoparticles leads to novel phenomena mediated by surface plasmon excitations. In this article we use single molecules to characterize the interaction of surface plasmons with light, and show that such interaction can strongly modulate the polarization of the emitted light. The simplest nanostructures that enable such polarization modulation are asymmetric silver nanocrystal trimers, where individual Raman scattering molecules are located in the gap between two of the nanoparticles. The third particle breaks the dipolar symmetry of the two-particle junction, generating a wavelength-dependent polarization pattern. Indeed, the scattered light becomes elliptically polarized and its intensity pattern is rotated in the presence of the third particle. We use a combination of spectroscopic observations on single molecules, scanning electron microscope imaging, and generalized Mie theory calculations to provide a full picture of the effect of particles on the polarization of the emitted light. Furthermore, our theoretical analysis allows us to show that the observed phenomenon is very sensitive to the size of the trimer particles and their relative position, suggesting future means for precise control of light polarization on the nanoscale.