Thiol‐Click Based Polyglycerol Hydrogels as Biosensing Platform with In Situ Encapsulated Streptavidin Probes

An in situ streptavidin‐encapsulated hydrogel based on dendritic polyglycerol (dPG) which is functionalized with either an acrylate, allyl or acrylamide group and dithiolated polyethylene glycol (PEG) is constructed via a thiol‐click chemistry approach and is investigated for biosensing applications. The hydrogel platform is screened for the encapsulation and release efficiencies of the model protein streptavidin under varying physicochemical conditions, for example, crosslinking chemistry reactions, the molar ratio between the two gel components, macromonomer concentrations or pH‐values. By that, tailor‐made hydrogels can be developed, which are able to encapsulate or release the model protein for several days based on its modality. Furthermore, the accessible binding site of encapsulated streptavidin or in other words, the biotin‐binding performance is quantified, and the stability of the various hydrogel types is studied by rheology measurements, 1H NMR, gel permeation chromatography (GPC), and mass loss experiments

Precise supramolecular control of surface coverage densities on polymer micro- and nanoparticles

We report herein the controlled surface functionalization of micro- and nanoparticles by supramolecular host–guest interactions. Our idea is to exploit the competition of two high-affinity guests for binding to the surface-bound supramolecular host cucurbit[7]uril (CB7). To establish our strategy, surface azide groups were introduced to hard-sphere (poly)methylmethacrylate particles with a grafted layer of poly(acrylic acid), and a propargyl derivative of CB7 was coupled to the surface by click chemistry. The amount of surface-bound CB7 was quantified with the high-affinity guest aminomethyladamantane (AMADA), which revealed CB7 surface coverage densities around 0.3 nmol cm 2 indicative of a 3D layer of CB7 binding sites on the surface. The potential for surface functionalization was demonstrated with an aminoadamantane-labeled rhodamine (Ada-Rho) as a second high-affinity guest. Simultaneous incubation of CB7-functionalized particles with both high-affinity guests, AMADA and Ada-Rho, revealed a simple linear relationship between the resulting surface coverage densities of the model fluorescent dye and the mole fraction of Ada-Rho in the incubation mixture. This suggests a highly modular supramolecular strategy for the stable immobilization of application-relevant molecules on particle surfaces and a precise control of their surface coverage densities.The financial support from the Deutsche Forschungsgemeinschaft (HE 5967/4-1 and NA 686/11-1), the China Scholarship Council (S. Z.), the Ministerio de Economia, Industria y Competitividad, Madrid (grant CTQ2014-54729-C2-1-P and predoctoral contract BES-2015-074458 for Z. D.), and the Ministerio de Ciencia, Innovacion y Universidades, Madrid (grant CTQ2017-89832-P) is gratefully acknowledged

Bioorthogonal in Situ Hydrogels Based on Polyether Polyols for New Biosensor Materials with High Sensitivity

Both noncovalent and covalent encapsulations of active biomolecules, for example, proteins and oligonucleotides, for a new biosensor matrix in an in situ bioorthogonal hydrogel formation via a strain-promoted azide–alkyne cycloaddition reaction were investigated. Unspecific interaction between the gel and the biomolecules as well as protein denaturation was prevented by the bioorthogonal gel components, which ensure a uniform aqueous environment in the hydrogel network. No leaching of the active biomolecules was observed. Additionally, a much higher and also adjustable loading of biomolecules in the hydrogel matrix was achieved compared to conventional biosensor surfaces, where the sensor molecules are immobilized on monolayers (2D surfaces) or brushlike structures (3D surfaces). Spotting experiments of the hydrogel confirm the possibility to use this new surface for microarray-based multiplex applications which require very high signal-to-noise ratios

Close Spectroscopic Look at Dye-Stained Polymer Microbeads

Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all parameters that can affect the fluorescence properties of the dye-stained beads

Scope and Limitations of Surface Functional Group Quantification Methods: Exploratory Study with Poly(acrylic acid)-Grafted Micro- and Nanoparticles

The amount of grafted poly­(acrylic acid) on poly­(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, ¹³C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]­uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly­(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network

Politicizing Europe: the challenge of executive discretion

Political decision-making in the Euro-crisis has relied heavily on executive discretion, exercised at speed and rationalised with reference to the pressing demands of emergency. This paper explores the challenges raised for political opposition, notably challenges of a temporal kind. With its deviations from policy and procedural norms, discretionary politics tends towards a politics without rhythm, leading to major asymmetries between decisionmakers and voices of opposition. These centre on issues of timing and the ability to identify authorship and content of decisions. Such asymmetries arguably correspond to an underlying one between the temporality of political decision-making and of contemporary finance capitalism, with agents of the former increasingly inclined to pursue ‘fast policy’ as a means to keep pace. A democratic response is likely to involve strengthening and synchronising the rhythms of parliamentary politics, as well as being receptive to forms of opposition less reliant on the rhythms that discretion subverts