Microfluidics allows the miniaturization of biochemical analyses. Small dimensions reduce sample and reagent consumption and enhance reaction rates. A downside is that high surface-to-volume ratios increase the unspecific binding of proteins to the substrate material. The resulting sample loss and reagent depletion decrease the sensitivity and specificity of protein-based assays, especially if low concentrations are analyzed. Here, we introduce the hydrophobin coating of microfluidic chips made of cyclic olefin copolymers (COC). The recombinant hydrophobin H*Protein B self-assembles into stable monolayers on hydrophobic surfaces, making them hydrophilic and thus reducing hydrophobic interactions between the chip surfaces and proteins. The substrate and sealing layers of the microfluidic chip were simply dip-coated and subsequently assembled by thermodiffusion bonding, which renders our coating procedure compatible with mass fabrication. Contact angle measurements and atomic force microscopy were used to evaluate the effect of high temperatures (107 degrees C) on COC substrates coated with H*Protein B. The efficiency of the protein-repellent coating was evaluated by depletion experiments with bovine serum albumin, human serum, and cerebrospinal fluid in microfluidic chips. Protein recovery was investigated down to protein concentrations of 0.3 mu g/mL. Recoveries of 90% were observed with total protein amounts of 10 ng, even for microfluidic channels up to 835 mm in length and with a cross section of 80 mu m x 230 mu m in a COC 6013/8007 foil. For comparison, only 30 and 60% of the protein was recovered in uncoated microfluidic channels with lengths of 835 and 128 mm, respectively. The long-term stability of the hydrophobin-coated chips for 8 weeks was demonstrated.

Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

Hydrophobins are highly surface active proteins which self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. We investigate hydrophobin self-assembly at oil/water interfaces to deepen the understanding of protein behavior in order to improve our biomimetic synthesis. Therefore, we carried out pendant drop measurements of hydrophobin stabilized oil/water systems determining the time-dependent IFT and the dilatational rheology with additional adaptation to the Serrien protein model. We show that the class I hydrophobin H(∗)Protein B adsorbs at an oil/water interface where it forms a densely-packed interfacial protein layer, which dissipates energy during droplet oscillation. Furthermore, the interfacial protein layer exhibits shear thinning behavior.

Background: Endoscopic stenting of the bile duct is a standard procedure for almost 35 years. In the case of long-term stenting occlusion of the stent is a major concern. Therefore optimizing biliary stents with respect to their patency is of great importance. We tested in an in animal study if coating of self-expanding metal stents with hydrophobin alone or hydrophobin with heparin reduces stent clogging as there were promising results in an in vitro study with this approach.Material and methods: In a randomized prospective animal study we implanted self-expanding metal stents either native or coated with hydrophobin alone or coated with hydrophobin and heparin into the bile duct of 15 pigs. After a survival period of 6 weeks we measured which part of the stent surface (%) was covered with clogging material using a commercially available image editing program on scanning electron microscopic images.Results: We found no differences between the native self-expanding metals stents and those coated with hydrophobin alone or hydrophobin and heparin.Conclusion: There are important differences in the clogging process between in vitro and in vivo models. Coating with hydrophobin with or without heparin is not able to inhibit the clogging process in an animal model.

Stent occlusion is a major complication in biliary stenting. This porcine study tested a newly developed coated biliary stent with a dedicated new surface proteine (hydrophobin) alone or with hydrophobin and heparin for preventing stent occlusion.

Die endoskopische Stentimplantation in den Gallengang ist eine Standardprozedur seit fast 35 Jahren. Die Einführung von selbstexpandierenden Metallstents war durch die Verlängerung der Offenheit ein wichtiger Fortschritt. Dennoch okkludieren auch selbstexpandierende Metallstents bei längerer Liegezeit. In einer vorausgehenden in vitro Untersuchung hatte sich gezeigt, dass eine Beschichtung von Stents mit Hydrophobin alleine oder zusammen mit Heparin die Adhärenz von Material an den Stent reduziert. In der vorliegenden Untersuchung sollte geprüft werden, ob dies auch in vivo der Fall ist.

Nature is capable of building materials with tailor-made properties under ambient conditions for specific applications. We apply some of the basic building principles of biomineralization in this paper: we stabilize an oil/water emulsion with the protein hydrophobin and mineralize this emulsion, resulting in hollow mineral capsules. The use of an emulsion as a liquid template enables precise size control over the final capsules. We mimic nature by using complexing agents and surfactants as additives to alter the properties of the growing mineral. We also modify the mineral itself by addition of different cations. Furthermore, we show the inclusion of silver into the capsules. This should add antibacterial properties to the capsules and shows exemplarily that catalytically active metals can be included. While the manual process needs numerous working steps and long waiting times, we ease the whole process by automation and use phosphatases to shorten synthesis time. Our experiments show the flexibility and adaptability of our system, making it an ideal platform for various possible applications such as drug transport and especially as microreactors.

L'invention concerne de nouveaux modulateurs du recepteur sensible au froid et au menthol TRPM8, des procedes destines a moduler le recepteur TRPM8 en utilisant lesdits modulateurs; et notamment l'utilisation desdits modulateurs pour induire une sensation de froid; ainsi que les objets et agents realises en utilisant lesdits modulateurs.

Hydrophobins, a class of highly surface active amphiphilic proteins, can stabilize various interfaces. When used for emulsion stabilization, it has been recently shown that they are able to induce mineralization, resulting in hollow capsules. We found that not all types of hydrophobins trigger mineralization, and that the morphology of the mineral changes depending on the selected oil. We investigated the formation of hydrophobin films at interfaces by the use of CD spectroscopy. In order to elucidate the structural features that enhance the mineralization property and give a possible explanation for this behavior, we performed MD-simulations of two representative hydrophobins (EAS for class I and HFBII for class II) at a hexane-water interface, in the presence as well as in the absence of ions. Our studies showed that the class II hydrophobin HFBII, which did not induce mineralization, only slightly changes its structure during adsorption at the oil-water interface or upon addition of Ca2+ and HPO42- ions. In contrast to that, the class I hydrophobin EAS changed its conformation to a large extent during the adsorption and interacted strongly with added ions. We revealed that EAS preorganizes the ions at short distances matching the lattice dimensions of hydroxyapatite. The latter finding yields a straightforward explanation for the observed differences in mineralization behavior and allows us to search for other hydrophobins that could assist mineralization, despite their different functions in nature.

By combining several surface analytical tools, we show that an adsorbed layer of the protein H*Protein B prevents the adsorption of secondary proteins bovine serum albumin, casein, or collagen at low-salinity conditions and at pH 8. H*Protein B is an industrially producible fusion protein of the hydrophobin family, known for its high interfacial activity. While applications of hydrophobin have been reported to facilitate adhesion of proteins under different pH conditions, careful analysis by quartz-crystal microbalance and ellipsometry prove that no additional adsorption can be found on top of the H*Protein B layer in this study. Surface analysis by X-ray photoelectron spectroscopy and secondary ion mass spectrometry proves that the hydrophobin layer stays intact even after hours of exposure to solutions of the secondary proteins and that no exchange of proteins can be detected.

Purpose: Although a wide range of biliary plastic and metal stents is on offer nowadays, the ideal cost-effective stent that functions permanently and that is easy to handle regarding its exchange is still not available. Therefore we tested in an in vitro model if the coating of plastic stents with hydrophobin alone or with hydrophobin and antibiotics or heparin in combination leads to an inhibition of the clogging process. Methods: We coated commercially available biliary plastic stents with hydrophobin alone, as well as with hydrophobin and antibiotics or heparin in combination. After an incubation period of 28 days in human bile, we examined the stents by scanning electron microscopy to see whether the clogging material on its surface was reduced. Results: Coating of plastic stents with hydrophobin led to a reduction in the amount of adherent material on the surface of the stents. Coupling of ampicillin/sulbactam or levofloxacin did not lead to a further reduction of the clogging material, whereas coupling with highly concentrated heparin did reduce the adherent material. Conclusions: The coating of biliary plastic stents with hydrophobin or with hydrophobin and heparin in combination seems to be a promising option to delay the clogging process.