Taylor Dispersion Analysis Research Articles

Size-Based Characterization of Polysaccharides by Taylor Dispersion Analysis with Photochemical Oxidation or Backscattering Interferometry Detections

Taylor dispersion analysis (TDA) is a powerful sizing technique very well suited for (macro)molecules between angstroms and submicrons (typically up to 200 nm). However, new detection modes are required for non-UV-absorbing (macro)molecules such as most of the polysaccharides, including starches. In this work, two different detection modes were compared, backscattering interferometry (BSI) and UV-photooxidation detection (UV-POD). TDA-BSI measures the relative change of the refractive index between eluent and sample (water as eluent in this work), whereas TDA-UV-POD detects the UV-absorbing photo-oxidized products of polysaccharides/starches in strong alkaline media (130 mM NaOH or 1 M KOH). TDA-UV-POD detection was evaluated for linearity and sensitivity at two wavelengths, 214 and 266 nm. The mass-average hydrodynamic radius (Rh) obtained by TDA-BSI and TDA-UV-POD was found to be in excellent agreement, whereas higher average Rh values were obtained by batch dynamic light scattering (DLS) under the same conditions, because of the higher sensitivity of DLS to large-size solutes and aggregates. The hydrodynamic radius distributions obtained by TDA and DLS are intrinsically different but both techniques were found to be complementary, providing useful information on sample dispersity. Owing to the absence of the stationary phase, low sample consumption with straightforward sample preparation (no filtration), and no calibration, TDA is anticipated to become a method of choice for the size-based characterization of polysaccharides, including starches.

Functionalized gold nanoparticles for sample preparation: A review.

Sample preparation is a crucial step for the reliable and accurate analysis of both small molecule and biopolymers which often involves processes such as isolation, pre-concentration, removal of interferences (purification), and pre-processing (e.g., enzymatic digestion) of targets from a complex matrix. Gold nanoparticle (GNP)-assisted sample preparation and pre-concentration has been extensively applied in many analytical procedures in recent years due to the favorable and unique properties of GNPs such as size-controlled synthesis, large surface-to-volume ratio, surface inertness, straightforward surface modification, easy separation requiring minimal manipulation of samples. This review article primarily focuses on applications of GNPs in sample preparation, in particular for bioaffinity capture and biocatalysis. In addition, their most common synthesis, surface modification and characterization methods are briefly summarized. Proper surface modification for GNPs designed in accordance to their target application directly influence their functionalities, e.g., extraction efficiencies, and catalytic efficiencies. Characterization of GNPs after synthesis and modification is worthwhile for monitoring and controlling the fabrication process to ensure proper quality and functionality. Parameters such as morphology, colloidal stability, and physical/chemical properties can be assessed by methods such as surface plasmon resonance, dynamic light scattering, ζ-potential determinations, transmission electron microscopy, Taylor dispersion analysis, and resonant mass measurement, among others. The accurate determination of the surface coverage appears to be also mandatory for the quality control of functionality of the nanoparticles. Some promising applications of (functionalized) GNPs for bioanalysis and sample preparation are described herein.

Electromembrane Extraction of Unconjugated Fluorescein Isothiocyanate from Solutions of Labeled Proteins Prior to Flow Induced Dispersion Analysis.

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 μL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 μL 0.1% (w/w) Aliquat 336 in 1-octanolas the supported liquid membrane (SLM) and 200 μL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (Kd) and hydrodynamic radius of the complex were determined to be 0.8 μM and 5.87 nm, respectively, which was in concordance with previously reported values.

Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

Supramolecular Self‐Assembly of DNA with a Cationic Polythiophene: From Polyplexes to Fibers

AbstractCationic polythiophenes constitute an interesting class of polymers for prospective applications in imaging, gene delivery and biosensing, as they combine solubility in aqueous media and have sensitive optical properties for the detection of biomolecules such as DNA. Here, we study the supramolecular self‐assembly of poly[3‐(6’‐(trimethylphosphonium)hexyl)thiophene‐2,5‐diyl] (P3HT‐PMe3) with different types of DNA such as single‐stranded oligonucleotides or long genomic DNA. Self‐assembly in buffered aqueous solution yields polyplexes (polymer/DNA complexes) with hydrodynamic radii ranging from 7 nm to around 25 nm, as observed by Taylor Dispersion Analysis. In these polyplexes, the achiral polymer presents chiroptical signals induced by supramolecular organization along chiral DNA. When the solution of long DNA/P3HT‐PMe3 is deposited on surfaces, tens of μm‐long fibers are formed by condensation of DNA and compaction of the polyplexes, as evidenced by microscopy techniques.

Peptide release from SEDDS containing hydrophobic ion pair therapeutic peptides measured by Taylor dispersion analysis

Therapeutic peptides are facing an increasing interest as drugs for the treatment of many diseases. The challenge in the administration of such drugs, due to inherent properties of these peptides, is to make them bioavailable. Self-emulsifying drug delivery systems (SEDDS) are considered a suitable and promising strategy to deliver the peptides and increase their bioavailability. However, to enter into the SEDDS nanodroplets, the peptides must be made hydrophobic by complexation with surfactants (formation of hydrophobic ion pair, HIP). The aim of this work is to assess the possibility to quantify the amount of released peptides and of the remaining docusate/peptide HIP in the nanodroplets by Taylor Dispersion Analysis (TDA) on two therapeutic peptides (leuprorelin and desmopressin). It also clearly demonstrates that the logP value of the peptide has a strong influence on the extent of HIP inside of the SEDDS nanodroplets. For instance leuprorelin-docusate complex (logP = 3) was 100% inside of the nanodroplets at low ionic strength, while for desmopressin-docusate complex (logP = 0.5) only 30% were able to enter the nanodroplets. It was also shown that an increase in the ionic strength of the release media allowed to increase the amount of released peptide up to 80% for leuprorelin and 100% for desmopressin, at physiological ionic strength. TDA experiments allowed to determine the partitioning coefficient, logD value, of the peptide between the SEDDS and continuous aqueous phases. In conclusion, this work demonstrates that TDA is a rapid, straightforward and useful technique for developing SEDDS formulations.

Flow-Induced Dispersion Analysis (FIDA) for Protein Quantification and Characterization.

Flow-Induced Dispersion Analysis (FIDA) enables characterization and quantification of proteins under native conditions. FIDA is based on measuring the change in size of a ligand as it selectively interacts with the target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), which increase in the presence of the analyte due to binding to the analyte. The Kd of the interaction may be obtained in a titration experiment and the measurement of the apparent ligand size in an unknown sample forms the basis for determining the analyte concentration. The apparent molecular size is measured by Taylor dispersion analysis (TDA) in fused silica capillary capillaries. FIDA is a "ligand-binding" assay and has therefore certain features in common with Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), and Biolayer Interferometry (BLI) based techniques. However, FIDA probes a single in-solution binding event and thus makes assay development straightforward, and the absolute size measurement enables built-in assay quality control. Further, as FIDA does not involve surface chemistries, complications related to nonspecific adsorption of analyte and assay components are minimized enabling direct measurement in, e.g., plasma and serum.

Structure and effective charge characterization of proteins by a mobility capillary electrophoresis based method.

Measuring the conformations and effective charges of proteins in solution is critical for investigating protein bioactivity, but their rapid analysis remains a challenging problem. Here we report a mobility capillary electrophoresis (MCE) based method for the rapid analysis of protein stereo-structures and effective charges in different solution environments. With the capability of mixture separation, MCE measures the hydrodynamic radius of a protein through Taylor dispersion analysis and its effective charge through ion mobility analysis. The experimental results acquired from MCE are then utilized to restrain molecular dynamics simulations, so that the most probable conformation of that protein can be obtained. As proof-of-concept demonstrations, the charge states and structures of five proteins were analyzed under close to native environments. The conformation transitions and charge state variations of bovine serum albumin and lysozyme under different pH conditions were also investigated. This method is promising for high-throughput protein analysis, which could potentially be coupled with mass spectrometry for investigating protein stereo-structures and functions in top-down proteomics.

Nanoparticles and Taylor Dispersion as a Linear Time-Invariant System.

The physical principles underpinning Taylor dispersion offer a high dynamic range to characterize the hydrodynamic radius of particles. While Taylor dispersion grants the ability to measure radius within nearly 5 orders of magnitude, the detection of particles is never instantaneous. It requires a finite sample volume, a finite detector area, and a finite detection time for measuring absorbance. First we show that these practical requirements bias the analysis when the self-diffusion coefficient of particles is high, which is typically the case of small nanoparticles. Second we show that the accuracy of the technique may be recovered by treating Taylor dispersion as a linear time-invariant system, which we prove by analyzing the Taylor dispersion spectra of two iron-oxide nanoparticles measured under identical experimental conditions. The consequence is that such treatment may be necessary whenever Taylor dispersion analysis is not optimized for a given size but dedicated to characterize broad groups of particles of varying size and material.

Heating behavior of magnetic iron oxide nanoparticles at clinically relevant concentration

Abstract Magnetic hyperthermia for cancer treatment has gained significant attention in recent years, due to its biocompatibility of applied nanoparticles and the possibility for spatially localized heating in deep tissues. Clinical treatments use nanoparticle concentrations of 112 mg Fe/mL, while the concentrations experimental studies have addressed are considerably smaller, usually between 0.1 and 30 mg/mL. Therefore, it is not clear whether such experiments correspond to the magnetic properties found in clinical applications. In this regard, we studied the thermal behavior of superparamagnetic iron oxide nanoparticles (SPION) with the most common particle shapes used in the field, including spherical (core diameters 11 and 19 nm), cubic (15 nm) and ellipsoidal (23 nm with an aspect ratio of 1.45), at concentrations ranging from 5 to 80 mg Fe/mL. Their shape, size, crystallinity, magnetic, and thermal behavior were characterized via transmission electron microscopy, dynamic light scattering, Taylor dispersion analysis, X-ray diffraction, alternating gradient magnetometry, and lock-in thermography. Spherical and cubic nanoparticles displayed linear heating slopes, independent from size, shape and concentration, resulting in unchanged specific absorption rates (SAR). Ellipsoids showed the same behavior until 50 mg/mL, above which a decreasing heating slope trend was found, without evidence as to what causes this behavior. However, the presented results highlight the importance of colloidally stable SPIONs in magnetic hyperthermia to obtain maximum heating power by minimum particle dosage.

Synthesis and characterization of polyampholytic aryl-sulfonated chitosans and their in vitro anticoagulant activity

This work firstly aimed to synthesize mono- and di- sulfonic derivatives of chitosan by reductive amination reaction using respectively 2-formyl benzene sulfonic acid and 2,4 formyl benzene sulfonic acid sodium salts. The influence of the reactants molar ratio (R), aryl – substituted amino groups versus chitosan free amino groups, on the degree of substitution (DS) of both sulfonated chitosans was assessed by 1H NMR, elemental analysis, coupled conductometry-potentiometry analysis and UV spectrometry and FTIR. The influence of pH on sulfonated chitosans’ properties in solution were investigated by solubility and zeta potential (ZP) studies, size exclusion chromatography equipped with MALLS detection (SEC-MALLS) and Taylor dispersion analysis (TDA). The polyampholytic character of both series was evidenced and strongly modified the solutions properties compared to chitosan. Then, the anticoagulant properties of mono- and di- sulfonic polymers were investigated by the measurement of the activated partial thromboplastin time (aPTT), Prothrombin-time (PT) and anti-(factor Xa).

Inline Coupling of Electrokinetic Preconcentration Method to Taylor Dispersion Analysis for Size-Based Characterization of Low-UV-Absorbing Nanoparticles.

The inline coupling of the field-amplified sample injection (FASI) to Taylor dispersion analysis (TDA) was used to characterize low-UV absorbing carboxylated silica nanoparticles (cNPs). The hydrodynamic diameters (Dh) were measured by using a commercial capillary electrophoresis instrument. The proposed methodology did not require any complicated instruments or chromophoric dye to increase the detection sensitivity. A practical method based on a half-Gaussian fitting was proposed for the data processing. The results obtained by this method were compared with those derived from dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. From these results, it appeared that the size derived by TDA is in excellent agreement with those measured by DLS and TEM, as demonstrated by stable nanoparticles with narrow size distributions. Intermediate precision relative standard deviations less than 5% were obtained by FASI-TDA. The effect of the FASI-induced cNP peak dispersion on the reliability of the results was discussed in detail.

Size-characterization of natural and synthetic polyisoprenes by Taylor dispersion analysis

Non-aqueous Taylor dispersion analysis (TDA) was used for the size-characterization of natural and synthetic polyisoprenes (4 × 103–2 × 106 g/mol molar mass). Not only the weight-average hydrodynamic radius (Rh), but also the probability distribution of the hydrodynamic radius, were both derived from the Taylorgrams by a simple integration of the elution profile and by a more sophisticated constrained regularized linear inversion of the Taylorgram, respectively. Results in terms of size characterization (hydrodynamic radii between 2 and 100 nm) were compared to size exclusion chromatography coupled to a refractive index-based mass detector. Multimodal size distributions were resolved by TDA for industrial and natural polyisoprenes, with the advantage over the chromatographic technique that, in TDA, there is no abnormal elution of microaggregates (hydrodynamic radii ∼ 40–50 nm). Considering the importance and the difficulty of characterizing polyisoprene microaggregates, TDA appears as a promising and simple technique for the characterization of synthetic and natural rubber.

Taylor Dispersion of Inorganic Nanoparticles and Comparison to Dynamic Light Scattering and Transmission Electron Microscopy

Taylor dispersion analysis (TDA) is an analytical method that has so far mainly been utilized to determine the diffusion coefficient of small molecules, and proteins. Due to increasing interest in nanoscience, some research has been done on the applicability of TDA towards characterizing nanoparticles. This work aims to expand this knowledge and give insight into the range for which TDA can be used for nanoparticle characterization, focusing on various materials and sizes. The TDA setup shown in this work was successful in characterizing all engineered metallic, non-metallic nanoparticles, and proteins tested in this work. Results were compared to dynamic light scattering and electron microscopy, and were in good agreement with both methods. Taking into consideration the wide range of nanoparticle sizes that can be characterized, the minimal sample preparation, and sample volume, required and the simplicity of the method, TDA can be considered as a valuable technique for nanoparticle characterization.

Size characterization of lipid-based self-emulsifying pharmaceutical excipients during lipolysis using Taylor dispersion analysis with fluorescence detection

Self-emulsifying drug delivery systems based on lipids have gained in interest in recent years due to their capacity to enhance the bioavailability of poorly water soluble drugs. Their oral intake suggests that they will be in contact with gastric and pancreatic enzymes during their passage through the gastrointestinal tract. The study of the evolution of such systems in the presence of enzymes is thus essential to develop better drug delivery vehicles. In this work, the lipolysis of two lipid based self-emulsifying drug delivery systems, Labrasol® and Gelucire® 44/14 by pancreatic enzymes and under conditions mimicking the gastrointestinal tract are presented. The following of the digestion is realized by Taylor dispersion analysis using fluorescent detection. A hydrophobic marker was used to tag the microdroplets. Results have shown that, Labrasol® droplets decrease exponentially in size with lipolysis time, from 11.8 nm to 3.5 nm in 20 min. On the contrary, Gelucire® 44/14 droplets increased sigmoïdally in size from 5.6 to 24.7 nm. Even after 120 min lipolysis, both systems maintained a solubilizing capacity of the hydrophobic marker.

Limits in Size of Taylor Dispersion Analysis: Representation of the Different Hydrodynamic Regimes and Application to the Size-Characterization of Cubosomes

Taylor dispersion analysis (TDA) is an absolute method (no calibration needed) for the determination of the molecular diffusion coefficient (D) based on the band broadening of a solute in a laminar flow. TDA is virtually applicable to any solute with size ranging from angstrom to sub-micrometer. The higher sizing limit is restricted by the occurrence of possibly two regimes: convective and hydrodynamic chromatography (HDC) regimes, which have different physical origins that should not be confused. This work aims at clearly defining the experimental conditions for which these two regimes can play a role, alone or concomitantly. It also calculates the relative error on D due to the HDC regime according to the solute to capillary size ratio. It is demonstrated in this work that HDC does not significantly affect the TDA measurement as long as the hydrodynamic radius of the solute is lower than 0.0051 times the capillary radius. Experimental illustrations of the occurrence of the two regimes are given taking polystyrene nanoparticles as model solutes. Finally, application of TDA to the sizing of large real-life solutes is proposed, taking cubosomes as new drug nanocarriers of potential interest for drug delivery purposes.

Taylor dispersion of nanoparticles

The ability to detect and accurately characterize particles is required by many fields of nanotechnology, including materials science, nanotoxicology, and nanomedicine. Among the most relevant physicochemical properties of nanoparticles, size and the related surface-to-volume ratio are fundamental ones. Taylor dispersion combines three independent phenomena to determine particle size: optical extinction, translational diffusion, and sheer-enhanced dispersion of nanoparticles subjected to a steady laminar flow. The interplay of these defines the apparent size. Considering that particles in fact are never truly uniform nor monodisperse, we rigorously address particle polydispersity and calculate the apparent particle size measured by Taylor dispersion analysis. We conducted case studies addressing aqueous suspensions of model particles and large-scale-produced “industrial” particles of both academic and commercial interest of various core materials and sizes, ranging from 15 to 100 nm. A comparison with particle sizes determined by transmission electron microscopy confirms that our approach is model-independent, non-parametric, and of general validity that provides an accurate account of size polydispersity—independently on the shape of the size distribution and without any assumption required a priori.

Taylor Dispersion Analysis of Polysaccharides Using Backscattering Interferometry.

Taylor dispersion analysis (TDA) allows the determination of the molecular diffusion coefficient (D) or the hydrodynamic radius (Rh) of a solute from the peak broadening of a plug of solute in a laminar Poiseuille flow. The main limitation plaguing the broader applicability of TDA is the lack of a sensitive detection modality. UV absorption is typically used with TDA but is only suitable for UV-absorbing or derivatized compounds. In this work, we present a development of the TDA method for non-UV absorbing compounds by using a universal detector based on refractive index (RI) sensing with backscattering interferometry (BSI). BSI was interfaced to a capillary electrophoresis-UV instrument using a polyimide coated fused silica capillary and an in-house designed flow-cell assembly. Polysaccharides were selected to demonstrate the application of TDA-BSI for size characterization. Under the conditions of validity of TDA, D and Rh average values and the entire Rh distributions were obtained from the (poly)saccharide taylorgrams, including non-UV absorbing polymers.

Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

We showed that the Taylor Dispersion Analysis (TDA) is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS). A series of measurements at constant protein concentration (for transferrin was 1.9 x 10−5 M, for β- lactoglobulin was 7.6 x 10−5 M, and for insulin was 1.2 x 10−4 M) and varying SDS concentrations were carried out in the phosphate-buffered saline (PBS). The structural changes were analyzed based on the diffusion coefficients of the complexes formed at various surfactant concentrations. The concentration of surfactant was varied in the range from 1.2 x 10−4 M to 8.7 x 10−2 M. We determined the minimum concentration of the surfactant necessary to change the native conformation of the proteins. The minimal concentration of SDS for β-lactoglobulin and transferrin was 4.3 x 10−4 M and for insulin 2.3 x 10−4 M. To evaluate the TDA as a novel method for studying denaturation of proteins we also applied other methods i.e. electronic circular dichroism (ECD) and dynamic light scattering (DLS) to study the same phenomenon. The results obtained using these methods were in agreement with the results from TDA.

Back Cover: Rapid determination of the critical micelle concentration by Taylor dispersion analysis in capillaries using both direct and indirect detection

Back Cover: Rapid determination of the critical micelle concentration by Taylor dispersion analysis in capillaries using both direct and indirect detection