Julien C. Gigault

Rational strategy for characterization of nanoscale particles by asymmetric-flow field flow fractionation: A tutorial

This tutorial proposes a comprehensive and rational measurement strategy that provides specific guidance for the application of asymmetric-flow field flow fractionation (A4F) to the size-dependent separation and characterization of nanoscale particles (NPs) dispersed in aqueous media. A range of fractionation conditions are considered, and challenging applications, including industrially relevant materials (e.g., metal NPs, asymmetric NPs), are utilized in order to validate and illustrate this approach. We demonstrate that optimization is material dependent and that polystyrene NPs, widely used as a reference standard for retention calibration in A4F, in fact represent a class of materials with unique selectivity, recovery and optimal conditions for fractionation; thus use of these standards to calibrate retention for other materials must be validated a posteriori. We discuss the use and relevance of different detection modalities that can potentially yield multi-dimensional and complementary information on NP systems. We illustrate the fractionation of atomically precise nanoclusters, which are the lower limit of the nanoscale regime. Conversely, we address the upper size limit for normal mode elution in A4F. The protocol for A4F fractionation, including the methods described in the present work is proposed as a standardized strategy to realize interlaboratory comparability and to facilitate the selection and validation of material-specific measurement parameters and conditions. It is intended for both novice and advanced users of this measurement technology.

Discriminating the states of matter in metallic nanoparticle transformations: what are we missing?

A limiting factor in assessing the risk of current and emerging nanomaterials in biological and environmental systems is the ability to accurately detect and characterize their size, shape, and composition in broad product distributions and complex media. Asymmetric flow field-flow fractionation (A4F) is capable of separation without stationary phase interactions or large applied forces. Here, we demonstrate unprecedented A4F fractionation of metallic nanoclusters with core diameters near 1 nm and with high resolution. The isolated nanocluster populations were characterized online with UV-vis absorption and inductively coupled plasma mass spectrometry (ICP-MS). We apply our methodology to a model system, poly(N-vinyl-2-pyrrolidone)-protected silver nanoparticles with an excess of tripeptide-glutathione (GSH). The temporal evolution of the initial silver nanoparticle distribution in the presence of excess GSH results in the appearance and persistence of a continuum of matter states (e.g., Ag(+) nanoclusters and nanoparticles) that could be fractionated with A4F, characterized by their optical signatures and diffusion coefficients, and quantified with ICP-MS. The results suggest that our methodology is generally applicable to metallic systems when appropriate online detection is coupled to the A4F. Because we extend the capability of the coupled A4F system to reliably detect, characterize, and quantify metallic populations in the sub-5 nm regime, the opportunity exists to survey the formation and transformation products of nanomaterials in more relevant biological and environmental systems. Thus, individually assessing the risks associated with specific ion, nanocluster, and nanoparticle populations is achievable, where such populations may have previously been misrepresented.

Observation of size-independent effects in nanoparticle retention behavior during asymmetric-flow field-flow fractionation

In this work, we highlight the size-independent influence of the material properties of nanoparticles (NPs) on their retention behavior in asymmetric-flow field-flow fractionation (A4F) by comparing four NP populations with similar nominal size. The phenomena described here suggest there are limits to the effectiveness and accuracy of using a single type of NP standard (polystyrene beads most typically) in order to generically calibrate retention time in normal mode elution. The dual objectives of this paper are to (1) demonstrate the uncertainties resulting from current practice and (2) initiate a discussion of these effects and their origins. The results presented here illustrate clearly that the retention time is higher for metallic NPs relative to lower (bulk) density NPs. By modifying the fundamental field-flow fractionation equation to account for differences in particle density, we show that the effect of the gravitational force is finite but insignificant for NPs. We postulate that the observed material-dependent retention behavior may be attributed to differences in the attractive van der Waals force between the NPs and the accumulation wall (membrane surface). We hope that our results will stimulate discussion and reassessment of the calibration procedure, perhaps by more fully accounting for all influential material parameters relevant to the fractionation of nanoscale particles by A4F.

Differentiation and characterization of isotopically modified silver nanoparticles in aqueous media using asymmetric-flow field flow fractionation coupled to optical detection and mass spectrometry.

The principal objective of this work was to develop and demonstrate a new methodology for silver nanoparticle (AgNP) detection and characterization based on asymmetric-flow field flow fractionation (A4F) coupled on-line to multiple detectors and using stable isotopes of Ag. This analytical approach opens the door to address many relevant scientific challenges concerning the transport and fate of nanomaterials in natural systems. We show that A4F must be optimized in order to effectively fractionate AgNPs and larger colloidal Ag particles. With the optimized method one can accurately determine the size, stability and optical properties of AgNPs and their agglomerates under variable conditions. In this investigation, we couple A4F to optical absorbance (UV-vis spectrometer) and scattering detectors (static and dynamic) and to an inductively coupled plasma mass spectrometer. With this combination of detection modes it is possible to determine the mass isotopic signature of AgNPs as a function of their size and optical properties, providing specificity necessary for tracing and differentiating labeled AgNPs from their naturally occurring or anthropogenic analogs. The methodology was then applied to standard estuarine sediment by doping the suspension with a known quantity of isotopically enriched (109)AgNPs stabilized by natural organic matter (standard humic and fulvic acids). The mass signature of the isotopically enriched AgNPs was recorded as a function of the measured particle size. We observed that AgNPs interact with different particulate components of the sediment, and also self-associate to form agglomerates in this model estuarine system. This work should have substantial ramifications for research concerning the environmental and biological fate of AgNPs.

Gold nanorod separation and characterization by asymmetric-flow field flow fractionation with UV–Vis detection

The application of asymmetric-flow field flow fractionation (A4F) for low aspect ratio gold nanorod (GNR) fractionation and characterization was comprehensively investigated. We report on two novel aspects of this application. The first addresses the analytical challenge involved in the fractionation of positively charged nanoparticles by A4F, due to the interaction that exists between the negatively charged native membrane and the analyte. We show that the mobile phase composition is a critical parameter for controlling fractionation and mitigating the membrane-analyte interaction. A mixture of ammonium nitrate and cetyl trimethyl ammonium bromide at different molar ratios enables separation of GNRs with high recovery. The second aspect is the demonstration of shape-based separation of GNRs in A4F normal mode elution (i.e., Brownian mode). We show that the elution of GNRs is due both to aspect ratio and a steric-entropic contribution for GNRs with the same diameter. This latter effect can be explained by their orientation vector inside the A4F channel. Our experimental results demonstrate the relevance of the theory described by Beckett and Giddings for non-spherical fractionation (Beckett and Giddings, J Colloid and Interface Sci 186(1):53–59, 1997). However, it is shown that this theory has its limit in the case of complex GNR mixtures, and that shape (i.e., aspect ratio) is the principal material parameter controlling elution of GNRs in A4F; the apparent translational diffusion coefficient of GNRs increases with aspect ratio. Finally, the performance of the methodology developed in this work is evaluated by the fractionation and characterization of individual components from a mixture of GNR aspect ratios.

Highly Stable Positively Charged Dendron-Encapsulated Gold Nanoparticles

We report the development of a novel cationic dendron (TAG1-PCD) and a positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering the reactivity, hydrophilicity, and cationic nature that is required to yield a stable gold conjugate in aqueous media. The PCD-AuNPs, nominally 10 nm in size, were synthesized by reduction of chloroauric acid in the presence of TAG1-PCD. The physicochemical properties of PCD-AuNPs were characterized by dynamic light scattering, transmission electron microscopy, UV-vis absorbance, and X-ray photoelectron spectroscopy for investigation of size distribution, shape uniformity, surface plasmon resonance bands, and Au-dendron bonding. Asymmetric-flow field flow fractionation was employed to confirm the in situ size, purity, and surface properties of the PCD-AuNPs. Additionally, the stability of PCD-AuNPs was systematically evaluated with respect to shelf life determination, stability in biological media and a wide range of pH values, chemical resistance against cyanide, redispersibility from lyophilized state, and stability at temperatures relevant to biological systems. Dose dependent cell viability was evaluated in vitro using the human lung epithelial cell line A549 and a monkey kidney Vero cell line. Observations from in vitro studies are discussed. Overall, the investigation confirmed the successful development of stable PCD-AuNPs with excellent stability in biologically relevant test media containing proteins and electrolytes, and with a shelf life exceeding 6 months. The excellent aqueous stability and apparent lack of toxicity for this conjugate enhances its potential use as a test material for investigating interactions between positively charged NPs and biocellular and biomolecular systems, or as a vehicle for drug delivery.

Quantitative analysis of dendron-conjugated cisplatin-complexed gold nanoparticles using scanning particle mobility mass spectrometry

We report a high-resolution and traceable method to quantify the drug loading on nanoparticle-based cancer therapeutics, and demonstrate this method using a model cisplatin functionalized dendron-gold nanoparticle (AuNP) conjugate. Electrospray differential mobility analysis (ES-DMA) provides upstream size classification based on the electrical mobility of AuNP conjugates in aerosol form following electrospray conversion from the aqueous suspension. A condensation particle counter (CPC) and inductively coupled plasma mass spectrometer (ICP-MS) provide the principal downstream quantification. CPC and ICP-MS yield complementary number-based and elemental mass-based particle size distributions, respectively. Conjugation using three different dendron formulations was differentiated based on changes in the mean mobility particle size. The subsequent cisplatin complexation to the dendron conjugates was quantified by coupling ES-DMA with ICP-MS. Discrete AuNP clusters (e.g., dimers, trimers) could be resolved from the relative quantity of atoms (i.e., Au and Pt) per particle after separation by ES-DMA. Surface density of cisplatin on Au was shown to be proportional to the density of carboxylic groups present and was independent of the state of AuNP clustering. Additionally, we found that colloidal stability of the conjugate is inversely proportional to the surface loading of cisplatin. This study demonstrates a prototype methodology to provide traceable quantification and to determine other important formulation factors relevant to therapeutic performance.

Saponins: A Renewable and Biodegradable Surfactant From Its Microwave-Assisted Extraction to the Synthesis of Monodisperse Lattices

Synthetic surfactants are widely used in emulsion polymerization, but it is increasingly desirable to replace them with naturally derived molecules with a reduced environmental burden. This study demonstrates the use of saponins as biodegradable and renewable surfactants for emulsion polymerization. This chemical has been extracted from soapnuts by microwave assisted extraction and characterized in terms of surfactant properties prior to emulsion polymerization. The results in terms of particle size distribution and morphology control have been compared to those obtained with classical nonionic (NP40) or anionic (SDS) industrial surfactants. Microwave-extracted saponins were able to lead to latexes as stable as standard PS latex, as shown by the CMC and CCC measurements. The saponin-stabilized PS particles have been characterized in terms of particle size and distribution by Dynamic Light Scattering and Asymmetrical Flow Field Flow Fractionation. Monomodal and monodispersed particles ranging from 250 to 480 nm in terms of diameter with a particle size distribution below 1.03 have been synthesized.

PEGylated gold nanorod separation based on aspect ratio: characterization by asymmetric-flow field flow fractionation with UV-Vis detection

AbstractThe development of highly efficient asymmetric-flow field flow fractionation (A4F) methodology for biocompatible PEGylated gold nanorods (GNR) without the need for surfactants in the mobile phase is presented. We report on the potential of A4F for rapid separation by evaluating the efficiency of functionalized surface coverage in terms of fractionation, retention time (tR) shifts, and population analysis. By optimizing the fractionation conditions, we observed that the mechanism of separation for PEGylated GNRs by A4F is the same as that for CTAB stabilized GNRs (i.e., according to their AR) which confirms that the elution mechanism is not dependent on the surface charge of the analytes and/or the membrane. In addition, we demonstrated that A4F can distinguish different surface coverage populations of PEGylated GNRs. The data established that a change in Mw of the functional group and/or surface orientation can be detected and fractionated by A4F. The findings in this study provide the foundation for a complete separation and physicochemical analysis of GNRs and their surface coatings, which can provide accurate and reproducible characterization critical to advancing biomedical research.n FigureA4F separation and elution of PEGylated gold nanorods (GNRs) are based on aspect ratio

Unexpected Changes in Functionality and Surface Coverage for Au Nanoparticle PEI Conjugates: Implications for Stability and Efficacy in Biological Systems

Cationic polyethylenimine conjugated gold nanoparticles (AuNP-PEI) are a widely studied vector for drug delivery and an effective probe for interrogating NP-cell interactions. However, an inconsistent body of literature currently exists regarding the reproducibility of physicochemical properties, colloidal stability, and efficacy for these species. To address this gap, we systematically examined the preparation, stability, and formation mechanism of PEI conjugates produced from citrate-capped AuNPs. We considered the dependence on relative molar mass, Mr, backbone conformation, and material source. The conjugation mechanism of Au-PEI was probed using attenuated total reflectance FTIR and X-ray photoelectron spectroscopy, revealing distinct fates for citrate when interacting with different PEI species. The differences in residual citrate, PEI properties, and sample preparation resulted in distinct products with differentiated stability. Overall, branched PEI (25 kDa) conjugates exhibited the greatest colloidal stability in all media tested. By contrast, linear PEI (25 kDa) induced agglomeration. Colloidal stability of the products was also observed to correlate with displaced citrate, which supports a glaring knowledge gap that has emerged regarding the role of this commonly used carboxylate species as a place holder for conjugation with ligands of broad functionalities. We observed an unexpected and previously unreported conversion of amine functional groups to quaternary ammonium species for 10 kDa branched conjugates. Results suggest that the AuNP surface catalyzes this conversion. The product is known to manifest distinct processes and uptake in biological systems compared to amines and may lead to unintentional toxicological consequences or decreased efficacy as delivery vectors. Overall, comprehensive physicochemical characterization (tandem spectroscopy methods combined with physical measurements) of the conjugation process provides a methodology for elucidating the contributing factors of colloidal stability and chemical functionality that likely influence the previously reported variations in conjugate properties and biological response models.