Selective Quantification of Bacteria in Mixtures by Using Glycosylated Polypyrrole/Hydrogel Nanolayers.

Here, we present a covalent nanolayer system that consists of a conductive and biorepulsive base layer topped by a layer carrying biorecognition sites. The layers are built up by electropolymerization of pyrrole derivatives that either carry polyglycerol brushes (for biorepulsivity) or glycoside moieties (as biorecognition sites). The polypyrrole backbone makes the resulting nanolayer systems conductive, opening the opportunity for constructing an electrochemistry-based sensor system. The basic concept of the sensor exploits the highly selective binding of carbohydrates by certain harmful bacteria, as bacterial adhesion and infection are a major threat to human health, and thus, a sensitive and selective detection of the respective bacteria by portable devices is highly desirable. To demonstrate the selectivity, two strains of Escherichia coli were selected. The first strain carries type 1 fimbriae, terminated by a lectin called FimH, which recognizes α-d-mannopyranosides, which is a carbohydrate that is commonly found on endothelial cells. The otherE. coli strain was of a strain that lacked this particular lectin. It could be demonstrated that hybrid nanolayer systems containing a very thin carbohydrate top layer (2 nm) show the highest discrimination (factor 80) between the different strains. Using electrochemical impedance spectroscopy, it was possible to quantify in vivo the type 1-fimbriated E. coli down to an optical density of OD600 = 0.0004 with a theoretical limit of 0.00005. Surprisingly, the selectivity and sensitivity of the sensing remained the same even in the presence of a large excess of nonbinding bacteria, making the system useful for the rapid and selective detection of pathogens in complex matrices. As the presented covalent nanolayer system is modularly built, it opens the opportunity to develop a broad band of mobile sensing devices suitable for various field applications such as bedside diagnostics or monitoring for bacterial contamination, e.g., in bioreactors.

Effect of the crosslinking agent on the biorepulsive and mechanical properties of polyglycerol membranes.

Polyglycerol (PG) based surfaces materials and surfaces are well-established bio-compatible materials. Crosslinking of the dendrimeric molecules via their OH groups improves their mechanical stability up to the point that free-standing materials can be attained. Here, we investigate the effect of different crosslinkers on PG films regarding their biorepulsivity and mechanical properties. For this purpose, PG films with different thicknesses (15, 50 and 100 nm) were prepared by polymerizing glycidol in a ring-opening polymerization onto hydroxyl-terminated Si substrates. These films were then crosslinked using ethylene glycol diglycidyl ether (EGDGE), divinyl sulfone (DVS), glutaraldehyde (GA), 1,11-di(mesyloxy)-3,6,9-trioxaundecane (TEG-Ms2) or 1,11-dibromo-3,6,9-trioxaundecane (TEG-Br2), respectively. While DVS, TEG-Ms2, and TEG-Br2 resulted in slightly thinned films, presumably due to loss of unbound material, increase of film thickness was observed with GA and, in particular, EDGDE, what can be explained by the different crosslinking mechanisms. The biorepulsive properties of the crosslinked PG films were characterized by water contact angle (WCA) goniometry and various adsorption assays involving proteins (serum albumine, fibrinogen, γ-globulin) and bacteria (E. coli), showing that some crosslinkers (EGDGE, DVS) improved the biorepulsive properties, while others deteriorated them (TEG-Ms2, TEG-Br2, GA). As the crosslinking stabilized the films, it was possible to use a lift-off procedure to obtain free-standing membranes if the thickness of the films was 50 nm or larger. Their mechanical properties were examined with a bulge test showing high elasticities, with the Young's moduli increasing in the order GA ≈ EDGDE < TEG-Br2 ≈ TEG-Ms2 < DVS.

Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy.

Transmission electron cryo-microscopy (cryoEM) of vitrified biological specimens is a powerful tool for structural biology. Current preparation of vitrified biological samples starts off with sample isolation and purification, followed by the fixation in a freestanding layer of amorphous ice. Here, we demonstrate that ultrathin (∼10 nm) smart molecular nanosheets having specific biorecognition sites embedded in a biorepulsive layer covalently bound to a mechanically stable carbon nanomembrane allow for a much simpler isolation and structural analysis. We characterize in detail the engineering of these nanosheets and their biorecognition properties employing complementary methods such as X-ray photoelectron and infrared spectroscopy, atomic force microscopy as well as surface plasmon resonance measurements. The desired functionality of the developed nanosheets is demonstrated by in situ selection of a His-tagged protein from a mixture and its subsequent structural analysis by cryoEM.

Self-Perforated Hydrogel Nanomembranes Facilitate Structural Analysis of Proteins by Electron Cryo-Microscopy.

We developed a method to improve specimen preparation for electron cryo-microscopy of membrane proteins. The method features a perforated hydrogel nanomembrane that stabilizes the thin film of aqueous buffer spanning the holes of holey carbon films, while at the same time preventing the depletion of protein molecules from these holes. The membrane is obtained by cross-linking of thiolated polyglycerol dendrimer films on gold, which self-perforate upon transfer to holey carbon substrates, forming a sub-micron-sized hydrogel network. The perforated nanomembrane improves the distribution of the protein molecules in the ice considerably. This facilitates data acquisition as demonstrated with two eukaryotic membrane protein complexes.