Methodological aspects on microdialysis protein sampling and quantification in biological fluids: an in vitro study on human ventricular CSF.

There is growing interest in sampling of protein biomarkers from the interstitial compartment of the brain and other organs using high molecular cutoff membrane microdialysis (MD) catheters. However, recent data suggest that protein sampling across such MD membranes is a highly complex process that needs to be further studied. Here, we report three major improvements for microdialysis sampling of proteins in complex biological matrixes.

Comparison of the adsorption kinetics and surface arrangement of "as received" and purified bovine submaxillary gland mucin (BSM) on hydrophilic surfaces.

The effect of bovine serum albumin (BSA) as impurity in a commercial bovine submaxillary gland mucin preparation (BSM; Sigma M3895) on the adsorption of BSM to hydrophilic surfaces (mica and silica) has been studied in terms of adsorption kinetics, amount and structure of the formed adlayer. The Surface Force Apparatus (SFA) was used to gain information about the extended and compressed structure of adsorbed "as received" BSM, purified BSM, BSA extracted from the "as received" BSM and mixtures of the latter purified proteins. The adsorbed amount was estimated using a combination of X-ray Photoelectron Spectroscopy (XPS), Enzyme-Linked Immuno Sorbent Assay (ELISA), Enzyme-Linked Lectin Assay (ELLA), Dual Polarization Interferometry (DPI) and Quartz Crystal Microbalance (QCM-D) measurements.

Surface analysis of pure and complex mucin coatings on a real-type substrate using individual and combined mBCA, ELLA, and ELISA.

In the past, we introduced the idea of using mucin coatings to improve biomaterials performance. Here, we evaluate non-radioactive methods for the analysis of pure and human host protein-containing (complex) mucin coatings on a real-type substrate (Thermanox). A common protein quantification assay (mBCA) was combined with mass-calibrated, enzyme-amplified assays based on lectin (ELLA) and antibody (ELISA) recognition, to determine the total and specific amounts of surface-associated proteins.

Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins.

Previously, we presented evidence that mucins have potential as biomaterial coatings. Here, we reveal substantial batch-to-batch variations for a frequently used commercial bovine salivary mucin preparation (BSM) and stress the importance of standardizing mucins intended for comparative purposes. "Mild" fractionation strategies, aiming at preserving natural mucin functions, were used to prepare two more defined BSM fractions as well as three mucin fractions from porcine gastric (PGM) and human salivary (MG1) sources.

Potential use of mucins as biomaterial coatings. II. Mucin coatings affect the conformation and neutrophil-activating properties of adsorbed host proteins--toward a mucosal mimic.

In continuation of our recent fractionation and characterization study on mucins of bovine salivary (BSM), porcine gastric (PGM), and human salivary (MG1) origin, this study evaluates the effect of mucin precoating on the conformation and neutrophil-activating properties of host proteins adsorbed to a polyethylene terephthalate-based model biomaterial. Microscopy combined with assays for the neutrophil releases of reactive oxygen species and human neutrophil lipocalin showed that mucin precoating greatly reduced the strong immune-response normally induced by adsorbed immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA), respectively. A similar finding was made for the proinflammatory fibrinogen.

Size characterization of inclusion bodies by sedimentation field-flow fractionation.

Sedimentation field-flow fractionation (sedFFF) was evaluated to characterize the size of Delta(4-23)TEM-beta-lactamase inclusion bodies (IBs) overexpressed in fed-batch cultivations of Escherichia coli. Heterologous Delta(4-23)TEM-beta-lactamase protein formed different sizes of IBs, depending upon the induction conditions. In the early phases of recombinant protein expression, induced with low concentrations of IPTG (isopropyl-beta-d-thiogalactoside), IB masses were larger than expected and showed heterogeneous size distributions.

Particulate platform for bioluminescent immunosensing.

The present study examines pyruvate kinase-conjugated antibodies for potential use in ELISA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles.

Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D).

The adsorption profile and viscoelastic properties of bovine submaxillary gland mucin (BSM) and bovine serum albumin (BSA), extracted from a commercial mucin preparation, adsorbing to polystyrene surfaces has been studied using quartz crystal microbalance with dissipation monitoring (QCM-D). A significant difference in the adsorption properties of the different proteins was detected; with the BSA adsorbing in a flat rigid layer whilst the mucin adsorbed in a diffuse, highly viscoelastic layer. Subsequent addition of BSA to the preadsorbed mucin layer resulted in stiffening of the protein layer which was attributed to complexation of the mucin by BSA.

Identification of cell adhesion molecules in the human follicle-associated epithelium that improve nanoparticle uptake into the Peyer's patches.

The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyer's patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker beta(1)-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence.

Nanoparticle decorated surfaces with potential use in glycosylation analysis.

A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range.

Characterization of surface-modified nanoparticles for in vivo biointeraction. A sedimentation field flow fractionation study.

Sedimentation field flow fractionation (SdFFF) is an emerging high-performance analytical tool for separation and determination of size and adsorption characteristics of colloidal particles. This study demonstrates how SdFFF can be used to characterize nanoparticles prepared for in vivo applications including (1) the quantification of polymer uptake on nanoparticles where surface coverage is crucial and (2) the coupling of cell adhesive peptides containing the Arg-Gly-Asp motif (RGD). Quantitative information about polymer adhesion in order to prepare a bioinert surface and an accurate determination of ligand uptake are both of obvious importance for the understanding of, for example, relations between the number of attached molecules for biointeraction and an observed therapeutic effect.

Shifts in polystyrene particle surface charge upon adsorption of the Pluronic F108 surfactant.

Electrical field-flow fractionation (ElFFF) and sedimentation field-flow fractionation (SdFFF) were used in combination to study the adsorption of the triblock polymeric surfactant, Pluronic F108 [(EO)129-(PO)56-(EO)129] to 200 nm polystyrene (PS) latex spheres. The SdFFF technique allowed an accurate determination of the mass of surfactant adsorbed on each particle from a solution of given concentration. To complement this isotherm study, we show that ElFFF can be used to measure fractional coverages of the formed electrically neutral surfactant layers on the charged PS particles.

Geometric scaling effects in electrical field flow fractionation. 2. Experimental results.

Geometric scaling of microelectrical field flow fractionation (micro-EFFF) systems is investigated experimentally and compared to theory and to macroscale EFFF systems. Experimental results are presented to demonstrate that the miniaturized system operates according to the scaling theory associated with the system. Demonstrated improvements in the channels include increased retention and resolution and decreased peak broadening, electrical time constants, relaxation time, power consumption, and sample size.