A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. Here we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. This high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

Characterisation of mass distributions of solvent-fractionated lignins using analytical ultracentrifugation and size exclusion chromatography methods

Lignins are valuable renewable resources for the potential production of a large array of biofuels, aromatic chemicals and biopolymers. Yet native and industrial lignins are complex, highly branched and heterogenous macromolecules, properties that have to date often undermined their use as starting materials in lignin valorisation strategies. Reliable knowledge of weight average molar mass, conformation and polydispersity of lignin starting materials can be proven to be crucial to and improve the prospects for the success of such strategies. Here we evaluated the use of commonly-used size exclusion chromatography (SEC)—calibrated with polystyrene sulphonate standards—and under-used analytical ultracentrifugation—which does not require calibration—to characterise a series of lignin fractions sequentially extracted from soda and Kraft alkaline lignins using ethyl acetate, methyl ethyl ketone (MEK), methanol and acetone:water (fractions F01–F04, respectively). Absolute values of weight average molar mass (Mw) determined using sedimentation equilibrium in the analytical ultracentrifuge of (3.0 ± 0.1) kDa and (4.2 ± 0.2) kDa for soda and Kraft lignins respectively, agreed closely with previous SEC-determined Mws and reasonably with the size exclusion chromatography measurements employed here, confirming the appropriateness of the standards (with the possible exceptions of fraction F05 for soda P1000 and F03 for Indulin). Both methods revealed the presence of low (~ 1 kDa) Mw material in F01 and F02 fractions followed by progressively higher Mw in subsequent fractions. Compositional analysis confirmed > 90% (by weight) total lignins successively extracted from both lignins using MEK, methanol and acetone:water (F02 to F04). Considerable heterogeneity of both unfractionated and fractionated lignins was revealed through determinations of both sedimentation coefficient distributions and polydispersity indices. The study also demonstrates the advantages of using analytical ultracentrifugation, both alongside SEC as well as in its own right, for determining absolute Mw, heterogeneity and conformation information for characterising industrial lignins.

Direct Measurement of Sedimentation Coefficient Distributions in Multimodal Nanoparticle Mixtures

Differential centrifugal sedimentation (DCS) is based on physical separation of nanoparticles in a centrifugal field prior to their analysis. It is suitable for resolving particle populations, which only slightly differ in size or density. Agglomeration presents a common problem in many natural and engineered processes. Reliable data on the agglomeration state are also crucial for hazard and risk assessment of nanomaterials and for grouping and read-across of nanoforms. Agglomeration results in polydisperse mixtures of nanoparticle clusters with multimodal distributions in size, density, and shape. These key parameters affect the sedimentation coefficient, which is the actual physical quantity measured in DCS, although the method is better known for particle sizing. The conversion into a particle size distribution is, however, based on the assumption of spherical shapes. The latter disregards the influence of the actual shape on the sedimentation rate. Sizes obtained in this way refer to equivalent diameters of spheres that sediment at the same velocity. This problem can be circumvented by focusing on the sedimentation coefficient distribution of complex nanoparticle mixtures. Knowledge of the latter is essential to implement and optimize preparative centrifugal routines, enabling precise and efficient sorting of complex nanoparticle mixtures. The determination of sedimentation coefficient distributions by DCS is demonstrated based on supracolloidal assemblies, which are often referred to as “colloidal molecules”. The DCS results are compared with sedimentation coefficients obtained from hydrodynamic bead-shell modeling. Furthermore, the practical implementation of the analytical findings into preparative centrifugal separations is explored.

Quantifying the concentration dependence of sedimentation coefficients for globular macromolecules: a continuing age-old problem

This retrospective investigation has established that the early theoretical attempts to directly incorporate the consequences of radial dilution into expressions for variation of the sedimentation coefficient as a function of the loading concentration in sedimentation velocity experiments require concentration distributions exhibiting far greater precision than that achieved by the optical systems of past and current analytical ultracentrifuges. In terms of current methods of sedimentation coefficient measurement, until such improvement is made, the simplest procedure for quantifying linear s-c dependence (or linear concentration dependence of 1/s) for dilute systems therefore entails consideration of the sedimentation coefficient obtained by standard c(s), g*(s) or G(s) analysis) as an average parameter ($$ \overline{s} $$ s ¯ ) that pertains to the corresponding mean plateau concentration (following radial dilution) ($$ \overline{c} $$ c ¯ ) over the range of sedimentation velocity distributions used for the determination of $$ \overline{s} $$ s ¯ . The relation of this with current descriptions of the concentration dependence of the sedimentation and translational diffusion coefficients is considered, together with a suggestion for the necessary improvement in the optical system.

Effective hard-sphere model of diffusion in aqueous polymer solutions

An effective hard-sphere model of the diffusion and cross-diffusion of salt in unentangled polymer solutions is developed. Given the viscosity, sedimentation coefficient and osmotic pressure of the polymer, the model predicts the diffusion and cross-diffusion coefficients as functions of the polymer concentration and molecular weight. The results are compared with experimental data on NaCl diffusion in aqueous polyethylene glycol solutions, showing good agreement at polymer molecular weights up to 400\,g/L. At higher molecular weights the model becomes less accurate, likely because of the effects of entanglement. The tracer Fickian diffusivity can be written in the form of a Stokes-Einstein equation containing the solution viscosity. For NaCl diffusion in polyethylene glycol solutions, the Stokes-Einstein equation breaks down as the polymer size increases. Using Batchelor's viscous correction factor to determine an effective viscosity experienced by the salt ions within the polymer matrix leads to much closer agreement with experiment.

Effective hard-sphere model of diffusion in aqueous polymer solutions

An effective hard-sphere model of the diffusion and cross-diffusion of salt in unentangled polymer solutions is developed. Given the viscosity, sedimentation coefficient and osmotic pressure of the polymer, the model predicts the diffusion and cross-diffusion coefficients as functions of the polymer concentration and molecular weight. The results are compared with experimental data on NaCl diffusion in aqueous polyethylene glycol solutions, showing good agreement at polymer molecular weights up to 400 g/L. At higher molecular weights the model becomes less accurate, likely because of the effects of entanglement. The tracer Fickian diffusivity can be written in the form of a Stokes-Einstein equation containing the solution viscosity. For NaCl diffusion in polyethylene glycol solutions, the Stokes-Einstein equation breaks down as the polymer size increases. Using Batchelor’s viscous correction factor to determine an effective viscosity experienced by the salt ions within the polymer matrix leads to much closer agreement with experiment.

Probing the effect of aroma compounds on the hydrodynamic properties of mucin glycoproteins

Aroma compounds are diverse low molecular weight organic molecules responsible for the flavour of food, medicines or cosmetics. Natural and artificial aroma compounds are manufactured and used by the industry to enhance the flavour and fragrance of products. While the low concentrations of aroma compounds present in food may leave no effect on the structural integrity of the mucosa, the effect of concentrated aroma volatiles is not well understood. At high concentrations, like those found in some flavoured products such as e-cigarettes, some aroma compounds are suggested to elicit a certain degree of change in the mucin glycoprotein network, depending on their functional group. These effects are particularly associated with carbonyl compounds such as aldehydes and ketones, but also phenols which may interact with mucin and other glycoproteins through other interaction mechanisms. This study demonstrates the formation of such interactions in vitro through the use of molecular hydrodynamics. Sedimentation velocity studies reveal that the strength of the carbonyl compound interaction is influenced by compound hydrophobicity, in which the more reactive short chain compounds show the largest increase in mucin-aroma sedimentation coefficients. By contrast, the presence of groups that increases the steric hindrance of the carbonyl group, such as ketones, produced a milder effect. The interaction effects were further demonstrated for hexanal using size exclusion chromatography light scattering (SEC-MALS) and intrinsic viscosity. In addition, phenolic aroma compounds were identified to reduce the sedimentation coefficient of mucin, which is consistent with interactions in the non-glycosylated mucin region.

Hydrodynamic Compatibility of Hyaluronic Acid and Tamarind Seed Polysaccharide as Ocular Mucin Supplements

Hyaluronic acid (HA) has been commonly used in eyedrop formulations due to its viscous lubricating properties even at low concentration, acting as a supplement for ocular mucin (principally MUC5AC) which diminishes with aging in a condition known as Keratoconjunctivitis sicca or “dry eye”. A difficulty has been its short residence time on ocular surfaces due to ocular clearance mechanisms which remove the polysaccharide almost immediately. To prolong its retention time, tamarind seed gum polysaccharide (TSP) is mixed as a helper biopolymer with HA. Here we look at the hydrodynamic characteristics of HA and TSP (weight average molar mass Mw and viscosity η) and then explore the compatibility of these polymers, including the possibility of potentially harmful aggregation effects. The research is based on a novel combination of three methods: sedimentation velocity in the analytical ultracentrifuge (SV-AUC), size-exclusion chromatography coupled to multiangle light scattering (SEC-MALS) and capillary viscometry. HA and TSP were found to have Mw=(680±30) kg/mol and (830±30) kg/mol respectively, and η=1475±30 ml/g and 675±20 ml/g, respectively. The structure of HA ranges from a rodlike molecule at lower molar masses changing to a random coil for Mw > 800 kg/mol, based on the Mark–Houwink–Kuhn–Sakurada (MHKS) coefficient. TSP, by contrast, is a random coil across the range of molar masses. For the mixed HA-TSP systems, SEC-MALS indicates a weak interaction. However, sedimentation coefficient (s) distributions obtained from SV-AUC measurements together with intrinsic viscosity demonstrated no evidence of any significant aggregation phenomenon, reassuring in terms of eye-drop formulation technology involving these substances.

Biological and physicochemical properties of cymbidium mosaic virus

The isolated cymbidium mosaic virus (CMV) is one of the most wide-spread and dangerous pathogens that infects promising varieties of orchids. It causes characteristic symptoms on orchid plants, which are manifested in the form of a mosaic. Over time, these areas are necrotized, leading to the stop of flowering the plants and reducing their decorative value. The CyMV is not spread by insects-carriers, but is transmitted by the mechanical inoculation with juice. Electron microscopy revealed flexible filamentous viral particles with a length of about 500 nm. The purified viral preparation is sedimented with a single peak with a sedimentation coefficient of 142S. The floating density of the virus in the preformed CsCl gradient corresponded to 1.3 g/cm3. The electrophoretic analysis of proteins in polyacrylamide gel under denatured conditions showed the presence of two polypeptides with molecular weights of 27 and 31 kDa. RNA CyMV has a molecular weight of 2 · 106 Da. In the translation system of rabbit reticulocytes in vitro, a protein with a molecular weight of about 27 kDa is synthesized. The obtained data allow us to refer CyMV to the group of potexviruses.

Modelling and Experimental Investigation on the Settling Rate of Kaolinite Particles in Non-Ideal Sedimentation Stage under Constant Gravity

We compared the catalytic effects of two polymers (soluble starch and apple pectin) on the flocculation of kaolinite suspension. Moreover, the relationship between the zeta potential value and the time when kaolin particle sedimentation occurred was verified, and the mechanism of flocculation was analyzed. Additionally, a constitutive model was proposed to simulate the non-ideal sedimentation of clay particles in an aqueous system under constant gravity. This model not only considers the inhomogeneity of the solute but also simulates the change in clay concentration with time during the deposition process. This model proposes a decay constant (α) and sedimentation coefficient (s). The model can also be used to calculate the instantaneous sedimentation rate of the clay suspensions at any time and any depth for the settling cylinder. These sedimentary characteristics were simulated by adopting the established deposition model. The results show that the model is capable of predicting the time required for the complete sedimentation of particles in the aqueous system, suggesting the feasibility of engineering wastewater treatment, site dredging, etc.