Taylor Dispersion Analysis Research Articles

Preventing the impact of solute adsorption in Taylor dispersion analysis: Application to protein and lipid nanoparticle analysis

Taylor Dispersion Analysis (TDA) allows diffusion coefficient (D) or hydrodynamic radius (Rh) determination on a wide range of size between angstroms and about 300 nm. However, solute adsorption phenomena can affect the repeatability and reproducibility of TDA. Several numerical studies addressed the theoretical impact of solute adsorption in TDA, but very few experimental studies focus on this topic and no experimental methodologies were proposed so far to reduce the impact of adsorption in TDA. In this work, an experimental protocol, called plug-in-front TDA, consisting of adding the solute in the eluent at a lower concentration compared to the injected sample, was proposed to strongly limit the impact of adsorption on the Rh determination. This protocol was suggested based on the evidence that adsorption / desorption phenomena impacting narrow bore fused silica TDA in aqueous conditions are typically slow processes that can be counteracted by saturating the interaction sites during the experiments. Successful applications to proteins and mRNA lipid nanoparticles (LNP) in vaccine against Covid 19 and protein analysis were reported. TDA of proteins in conditions of strong interactions with the capillary surface was possible using the plug-in-front methodology. We anticipate that such experimental methodology will greatly help the experimentalist for implementing TDA in various applications.

Simultaneously performing Taylor dispersion analysis with fluorimetry, photometry, and contactless conductometry at the same detection window

Taylor dispersion analysis (TDA) is a rapid and precise method for determining the hydrodynamic radius (RH) of various substances. We present a versatile TDA system with a flow-through sample injection device, two compact 3-in-1 detectors, and a high-voltage power supply. The 3D-printed detectors combine fluorimetry (FD), photometry (AD@255 nm), and contactless conductometry (C4D) in a single head, enabling simultaneous detection at one capillary window. Using bovine serum albumin (BSA) as a model analyte, we compare TDA with different detection methods. BSA labeled with fluorescein isothiocyanate (FITC) is analyzed in both pulse mode and capillary electrophoresis (CE) TDA. FD and AD detection yield similar RH values, except when FITC binds with small ions in the buffer. In phosphate buffer, C4D underestimates RH values by approximately 18 % due to BSA self-association. In Tris-based buffers, C4D values are 87%–96 % of AD values in pulse mode. With CE-TDA using Tris-CHES buffer, no statistical difference is found across all detections. The system is also applied to CE-TDA of various compounds, particularly charged saccharides. CE-TDA improves the accuracy of TDA results from C4D. We demonstrate the resolution of mixed C4D-TDA signals with assistance from FD and AD signals, successfully resolving gluconate peaks fully covered by another compound. The versatile system with 3-in-1 detection offers a powerful tool for TDA of mixtures and enhances sample throughput.

Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Structure-Activity Optimization of Luminescent Tb-doped LaF3 Nanoparticles.

A series of Tb-doped LaF3 nanoparticles (NPs) was prepared by systematically varying the Tb doping rate from 0 to 100%. The elemental composition was confirmed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis, and the size, morphology, and crystal structure were determined in the solid state by transmission electron microscopy and X-ray diffractometry, while the size and ζ-potential of the NPs in solution were studied by dynamic light scattering, Taylor dispersion analysis, and laser Doppler electrophoresis. While the crystal structure appears to be hexagonal for a doping rate of up to 70%, an admixture of hexagonal and orthorhombic phases is observed for 80 and 90% Tb contents with a pure orthorhombic phase being obtained for TbF3. The spectroscopic properties of the NPs were studied for bare NPs and in the presence of dipicolinic acid as a surface-capping antenna ligand in solution. The coverage of the NPs by the ligand resulted in an increase in the luminescence lifetime of the emitting Tb centers, as a consequence of a better protection toward luminescence quenching from water molecules, as well as a large improvement in the brightness of the NPs. Taking into account the various parameters, a doping rate of 40% Tb was shown to be the best compromise for the development of such NPs for bioanalytical applications.

Taylor dispersion analysis for measurement of diffusivity and size of gadolinium-based contrast agents

Gadolinium-based contrast agents (GBCA) are complexes of a Gadolinium metal center and a linear or macrocyclic polyamino-carboxylic acid chelating agent. These agents are employed to enhance the visibility of deep abnormalities through MRI techniques. Knowing the precise dimensions of various GBCA is key parameter for understanding their in-vivo and pharmaco-kinetic behaviors, their diffusivity, as well as their relaxivity. However, conventional size characterization techniques fall short when dealing with these tiny molecules (≤1 nm). In this work, we propose to determine the size and diffusivity of gadolinium-based contrast agents using Taylor dispersion analysis (TDA). TDA provided a reliable measurement of the hydrodynamic diameter and the diffusion coefficient. The obtained results were compared to DOSY NMR (Diffusion-ordered Nuclear Magnetic Resonance Spectroscopy) and DFT (Density Functional Theory).

Characterization of Nanoparticles in Mixtures by Taylor Dispersion Analysis Hyphenated to Inductively Coupled Plasma Mass Spectrometry.

A novel methodology for investigating the behavior of nanoparticles in their mixtures in aqueous high-ionic strength conditions is presented in this work. Our approach utilizes Taylor dispersion analysis in capillaries connected to inductively coupled plasma mass spectrometry (ICP-MS) to probe metal-derived nanoparticles. This methodology simultaneously distinguishes between different kinds of nanoparticles and accurately determines their essential parameters, such as hydrodynamic size, diffusion coefficient, and elemental composition. Moreover, the isotope-specific ICP-MS detection allows for unique targeting of the fate of isotopically enriched nanoparticles. The complexity of our methodology opens the way for studying barely explored areas of interparticle interactions or unequivocal characterization of one type of nanoparticle in complex mixtures without any need for calibration as well as labor-consuming sample preparation.

Numerical modeling and experimental optimization of Taylor dispersion analysis with and without an electric field.

Numerical modeling of Taylor dispersion analysis (TDA) was performed using COMSOL Multiphysics to facilitate better and faster optimization of the experimental conditions. Parameters, such as pressure, electric field, diameter, and length of capillary on the TDA conditions, were examined for particles with hydrodynamic radius (Rh) of 2.5-250Å. The simulations were conducted using 25, 50, and 100cm length tubes with diameters of 25, 50, and 100µm. It was shown that particles with larger diffusion coefficients gave more accurate results at higher velocities, and in longer and wider columns; particles with smaller diffusion coefficients gave more accurate results at smaller velocities, and in shorter and thinner columns. Moreover, the effect of electric field on the validity and the applicability of TDA was studied using TDA in conjunction with capillary electrophoresis. Diffusion coefficients were obtained using a pressure and the TDA equation and compared with those obtained with a pressure in combination of an electric field for fluorescein, FD4, FD20, FD70, and FD500. We found that TDA can be used with the presence of moderate electrophoretic migration and electroosmotic flow, when appropriate conditions were met.

Abstract 1939 High sensitivity analysis of tau aggregation models by capillary electrophoresis and Taylor dispersion analysis

Abstract 1939 High sensitivity analysis of tau aggregation models by capillary electrophoresis and Taylor dispersion analysis

Taylor dispersion analysis using capacitively coupled contactless conductivity detector

Taylor dispersion analysis (TDA) is a simple and absolute method to determine the hydrodynamic radius of solutes that respond to UV or fluorescence detections. To broaden the application range of TDA, it is necessary to develop new detection modes. This study aims to study capacitively coupled contactless conductivity detector (C4D) for the analysis of charged macromolecules. The detection sensitivities and hydrodynamic radii were compared for a C4D detector and a UV detector on positively or negatively charged polymers responding both to UV and C4D (poly-L-lysine and poly(acrylamide-co-2-acrylamido-1-methyl-propanesulfonate). The influence of the composition of the background electrolyte on the detection sensitivity has been studied and optimized for C4D detection. The influence of the molar mass and of the polymer chemical charge density on the C4D and UV sensitivities of detection have been investigated based on well-characterized copolymers samples of different molar masses and charge densities. The advantages and disadvantages compared to UV detection, as well as the range of applicability of C4D detection in TDA were identified. C4D detection can be an alternative method for sizing charged polymers of reasonable molar mass (typically below 105 g mol−1) that do not absorb in UV. A decline in the sensitivity of detection in C4D was observed for higher molar masses.

Polyglycerol fatty acid ester contributes to the improvement and maintenance of water solubility of amorphous curcumin by suppressing the intermolecular interaction and the diffusion rate of curcumin

Curcumin (CUR), a polyphenol, is an attractive component of functional foods, owing to various physiological activities. However, CUR is highly hydrophobic, insoluble in water, and difficult to absorb in the body. Here, we report an amorphous CUR formulation containing the dispersant polyglycerol fatty acid ester (PGFE), demonstrating high and stable water solubility. Improved water solubility enhanced the absorbability of CUR in our amorphous formulation along with enhanced triglyceride inhibition, compared to that in a commercial formulation. Nuclear Overhauser effect spectroscopy (NOESY) analysis revealed that PGFE reduced CUR-CUR interaction, resulting in higher dispersion and improved solubility of CUR. Taylor dispersion analysis showed a lower diffusion coefficient of CUR in the highly water-soluble formulation (with PGFE) than that in the low water-soluble formulation (without PGFE), which prevents recontact and recrystallization of CUR, which is trapped by PGFE. Overall, the amorphous CUR with high solubility could be used as a promising functional food.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30–1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120–210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1–13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Optimization of limit of detection in Taylor dispersion analysis: Application to the size determination of vaccine antigens

The development of a new vaccine requires the precise characterization of all the physicochemical parameters of the vaccine antigens, which are the molecules that induce the immune response. Taylor dispersion analysis (TDA) is a promising alternative technique for the determination of diffusion coefficients and hydrodynamic radii of proteins, macromolecules and nanoparticles. In this work, TDA was used to determine the hydrodynamic radius distribution and its average value of four antigens: diphtheria toxoid (DT), tetanus toxoid (TT), hepatitis B surface antigen (HBsAg) and polyribosyl-ribitol phosphate conjugated to tetanus toxoid (PRP-T). The robustness of the results obtained was investigated on bare fused silica capillary and hydroxypropylcellulose coated capillary. The impact of operational parameters on the limit of detection (LOD) and limit of quantification (LOQ) were studied from both theoretical and experimental points of view. The influence of the diameter and the length of the capillary on the LOD and LOQ were studied as well as the impact of the mobilization pressure. General guidelines for the choice of the initial operating conditions are given for the development of future TDA methods.

β-Cyclodextrin and Folic Acid Host-Guest Interaction Binding Parameters Determined by Taylor Dispersion Analysis and Affinity Capillary Electrophoresis.

The thermodynamic properties of molecular recognition in host-guest inclusion complexes can be studied by Taylor dispersion analysis (TDA). Host-guest inclusion complexes have modest size, and it is possible to get convergent results fast, achieving greater certainty for the obtained thermodynamic properties. Cyclodextrins (CDs) and their derivatives can be used as drug carriers that can boost stability, solubility, and bioavailability of physiologically active substances. A simple and effective approach for assessing the binding properties of CD complexesthat are critical in the early stages of drug and formulation development is needed to fully understand the process of CD and guest molecules' complex formation. In this work, TDA was successfully used to rapidly determine interaction parameters, including binding constant and stoichiometry, between β-CD and folic acid (FA) along with the diffusivities of the free FA and its complex with β-CD. Additionally, the FA diffusion coefficient obtained by TDA was compared to the results previously obtained by NMR. Affinity capillary electrophoresis (ACE) was also used to compare the binding constants obtained by different methods. The results showed that the binding constants obtained by ACE were somewhat lower than those obtained by the two TDA procedures. This article is protected by copyright. All rights reserved.

Cross-frontal mode: An alternative methodology for Taylor dispersion analysis of monomodal sample

Taylor dispersion analysis (TDA) is a technique dedicated to the determination of the molecular diffusion coefficient (D) of species, using band broadening of an analyte in a laminar flow. Two modes are commonly used to perform TDA: pulse and frontal modes. In each case, a fitting of the signal is required. We propose here a third mode denoted as cross-frontal mode, combining two crossed sample fronts without modification of a classical CE device for the rapid and accurate determination of D of caffeine, reduced glutathione (GSH), insulin from bovine pancreas, bovine serum albumin (BSA) and citrate-capped gold nanoparticles (AuNP). Theoretical aspects and methodology are described, showing a good correlation between the so-called cross-frontal mode and usual frontal mode. Limitations of the techniques are also assessed, and are similar to regular modes while no fitting is required. This new methodology allows improving the sensitivity toward low concentrated sample compared to pulse mode, and an alternative mathematical treatment compared to regular TDA modes.

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Taylor dispersion analysis discloses the impairment of Aβ peptide aggregation by the presence of a fluorescent tag.

The use of fluorescently tagged amyloid peptides, implicated in Alzheimer's disease, to study their aggregation at low concentrations is a common method; however, the fluorescent tag should not introduce a bias in the aggregation process. In this work, native amyloid peptides Aβ(1-40) and Aβ(1-42) and fluorescein-5-isothiocyanate (FITC), tagged ones, were studied using Taylor dispersion analysis coupled with a simultaneous UV and light-emitting diode-induced fluorescence detection, to unravel the effect of FITC on the aggregation process. For that, a total concentration of 100µM of peptides consisting of a mixture of native and tagged ones (up to 10% in moles) was applied. Results demonstrated that FITC had a strong inhibition effect upon the aggregation behaviour of Aβ(1-42), whereas for Aβ(1-40), only a retardation in kinetics was observed. It was also shown that when mixed solutions of Aβ(1-40) and Aβ(1-42) are used, the Aβ(1-42) alloform was the leading peptide in the aggregation process, and when the latter was tagged, the aggregation kinetics decreased but the lifetime of potentially toxic oligomers was drastically increased. These results confirmed that the hydrophilicity of the N-terminus part of the peptide plays a major role in the aggregation process.

Taylor Dispersion Analysis to support lipid-nanoparticle formulations for mRNA vaccines.

Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.

Taylor dispersion analysis of metallic-based nanoparticles - A short review.

Taylor dispersion analysis (TDA) is an interesting tool for nanoparticle (NP) size determination, feasible using simple capillary electrophoresis apparatus. Based upon the radial diffusion of analytes upon a laminar stream, the diffusion coefficient of species is easily estimable. Moreover, TDA is generally more adequate than conventional dynamic light scattering methodologies as it is less dependent on the polydispersity of the sample, leading to accurate measurement and reliable results. This review provides every paper mentioning the use of TDA for metallic-based NPs size determination. Diverse strategies for the detection of metallic NPs (like UV-visible and inductively coupled plasma-mass spectrometry - ICP-MS - for instance) and interpretation of the Taylorgrams are discussed. Based upon the literature, advices on future prospects are also indicated, especially for the comparison of TDA results with other classical techniques.

Diffusioosmosis-driven dispersion of colloids: a Taylor dispersion analysis with experimental validation

Diffusiophoresis refers to the movement of colloidal particles in the presence of a concentration gradient of a solute and enables directed motion of colloidal particles in geometries that are inaccessible, such as dead-end pores, without imposing an external field. Previous experimental reports on dead-end pore geometries show that, even in the absence of mean flow, colloidal particles moving through diffusiophoresis exhibit significant dispersion. Existing models of diffusiophoresis are not able to predict the dispersion and thus the comparison between the experiments and the models is largely qualitative. To address these quantitative differences between the experiments and models, we derive an effective one-dimensional equation, similar to a Taylor dispersion analysis, that accounts for the dispersion created by diffusioosmotic flow from the channel sidewalls. We derive the effective dispersion coefficient and validate our results by comparing them with direct numerical simulations. We also compare our model with experiments and obtain quantitative agreement for a wide range of colloidal particle sizes. Our analysis reveals two important conclusions. First, in the absence of mean flow, dispersion is driven by the flow created by diffusioosmotic wall slip such that spreading can be reduced by decreasing the channel wall diffusioosmotic mobility. Second, the model can explain the spreading of colloids in a dead-end pore for a wide range of particle sizes. We note that, while the analysis presented here focuses on a dead-end pore geometry with no mean flow, our theoretical framework is general and can be adapted to other geometries and other background flows.

3D Printed Instrument for Taylor Dispersion Analysis with Two-Point Laser-Induced Fluorescence Detection.

Precisely controlling the size of engineered biomolecules and pharmaceutical compounds is often critical to their function. Standard methods for size characterization, such as dynamic light scattering or size exclusion chromatography, can be sample intensive and may not provide the sensitivity needed for mass- or concentration-limited biological systems. Taylor dispersion analysis (TDA) is a proven analytical method for direct, calibration-free size determination which utilizes only nL-pL sample volumes. In TDA, diffusion coefficients, which are mathematically transformed to hydrodynamic radii, are determined by characterizing band broadening of an analyte under well-controlled laminar flow conditions. Here, we describe the design and development of a 3D printed instrument for TDA, which is the first such instrument to offer dual-point laser-induced fluorescence (LIF) detection. The instrument utilized a fully 3D printed eductor as a vacuum source for precise and stable pressure-driven flow within a capillary, evidenced by a linear response in generated static pressure to applied gas pressure (R2 = 0.997) and a 30-fold improvement in stability of static pressure (0.05% RSD) as compared to a standard mechanical pump (1.53%). Design aspects of the LIF detection system were optimized to maximize S/N for excitation and emission optical axes, and high sensitivity was achieved as evidenced by an 80 pM limit of detection for the protein R-Phycoerythrin and low nM limits of detection for three additional fluorophores. The utility of the instrument was demonstrated via sizing of R-Phycoerythrin at pM concentrations.