Polyethylene imine-based receptor immobilization for label free bioassays

Polyethylene imine (PEI) based immobilization of antibodies is described and the concept is proved on the label free assay of C-Reactive Protein (CRP). This novel immobilization method is composed of a hyperbranched PEI layer which was deposited at a high pH (9.5) on the sensor surface. The free amino groups of PEI were derivatized with neutravidin by Biotin N-hydroxysuccinimide ester and the biotinylated anti-CRP antibody immobilized on this layer. Direct binding assay of recombinant CRP was successfully performed in the low μg/ml concentrations using a label free optical waveguide biosensor

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels

Label-Free in Situ Optical Monitoring of the Adsorption of Oppositely Charged Metal Nanoparticles

The mechanism of alternating deposition of oppositely charged gold nanoparticles (AuNPs) was investigated by optical waveguide lightmode spectroscopy (OWLS). OWLS allows monitoring of the kinetics of layer-by-layer (LbL) adsorption of positively and negatively charged nanoparticles in real time without using any labels so that the dynamics of layer formation can be revealed. Positively charged NPs that are already deposited on a negatively charged glass substrate strongly facilitate the adsorption of the negatively charged particles. The morphology of the adsorbed layer was also investigated with atomic force microscopy (AFM). AFM revealed that the interaction between oppositely charged particles results in the formation of NP clusters with sizes varying between 100 and 6000 NPs. The cluster size distribution is found to be an exponentially decaying function, and we propose a simple theory to explain this finding

CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18.

Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) belong to the family of beta2 integrins and are expressed mainly by myeloid cell types in humans. Previously, we proved that CR3 rather than CR4 plays a key role in phagocytosis. Here we analysed how CD11b and CD11c participate in cell adhesion to fibrinogen, a common ligand of CR3 and CR4, employing human monocytes, monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) highly expressing CD11b as well as CD11c. We determined the exact numbers of CD11b and CD11c on these cell types by a bead-based technique, and found that the ratio of CD11b/CD11c is 1.2 for MDDCs, 1.7 for MDMs and 7.1 for monocytes, suggesting that the function of CD11c is preponderant in MDDCs and less pronounced in monocytes. Applying state-of-the-art biophysical techniques, we proved that cellular adherence to fibrinogen is dominated by CD11c. Furthermore, we found that blocking CD11b significantly enhances the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand. On the basis of the cell surface receptor numbers and the measured adhesion strength we set up a model, which explains the different behavior of the three cell types