Controlling Fluorescent Readout in Paper-based Analytical Devices.

Paper is an ideal candidate for the development of new disposable diagnostic devices because it is a low-cost material, allows transport of the liquid on the device by capillary action, and is environmentally friendly. Today, colorimetric analysis is most often used as a detection method for rapid tests (test strips or lateral flow devices) but usually gives only qualitative results and is limited by a relatively high detection threshold. Here, we describe studies using fluorescence as a readout tool for paper-based diagnostics. We study how the optical readout is affected by light transmission, scattering, and fluorescence as a function of paper characteristics such as thickness (grammage), water content, autofluorescence, and paper type/composition. We show that paper-based fluorescence analysis allows better optical readout compared to that of nitrocellulose, which is currently the material of choice in colorimetric assays. To reduce the loss of analyte molecules (e.g., proteins) due to adsorption to the paper surface, we coat the paper fibers with a protein-repellent hydrogel. For this purpose, we use hydrophilic copolymers consisting of N,N-dimethyl acrylamide and a benzophenone-based cross-linker, which are photochemically transformed into a fiber-attached polymer hydrogel on the paper fiber surfaces in situ. We show that the combination of fluorescence detection and the use of a protein-repellent coating enables sensitive paper-based analysis. Finally, the success of the strategy is demonstrated by using a simple LFD application as an example.

Novel Motion Sequences in Plant-Inspired Robotics: Combining Inspirations from Snap-Trapping in Two Plant Species into an Artificial Venus Flytrap Demonstrator.

The field of plant-inspired robotics is based on principles underlying the movements and attachment and adaptability strategies of plants, which together with their materials systems serve as concept generators. The transference of the functions and underlying structural principles of plants thus enables the development of novel life-like technical materials systems. For example, principles involved in the hinge-less movements of carnivorous snap-trap plants and climbing plants can be used in technical applications. A combination of the snap-trap motion of two plant species (Aldrovanda vesiculosa and Dionaea muscipula) has led to the creation of a novel motion sequence for plant-inspired robotics in an artificial Venus flytrap system, the Venus Flyflap. The novel motion pattern of Venus Flyflap lobes has been characterized by using four state-of-the-art actuation systems. A kinematic analysis of the individual phases of the new motion cycle has been performed by utilizing precise pneumatic actuation. Contactless magnetic actuation augments lobe motion into energy-efficient resonance-like oscillatory motion. The use of environmentally driven actuator materials has allowed autonomous motion generation via changes in environmental conditions. Measurement of the energy required for the differently actuated movements has shown that the Venus Flyflap is not only faster than the biological models in its closing movement, but also requires less energy in certain cases for the execution of this movement.

"CHicable" and "Clickable" Copolymers for Network Formation and Surface Modification.

In this study, we present the generation of novel, multifunctional polymer networks through a combination of C,H-insertion cross-linking (CHic) and click chemistry. To this, copolymers consisting of hydrophilic N,N-dimethylacrylamide as matrix component and repeat units containing azide moieties, as well as benzophenone or anthraquinone groups, are generated. The benzophenone or anthraquinone groups allow photo-cross-linking, surface attachment or covalent immobilization of adjacent (bio)molecules through CHic reactions. The azide moieties either can react with available alkynes through conventional click reactions or can be activated to form nitrenes, which can also undergo CHic reactions. By choosing appropriate reaction conditions, the same polymer can be used to follow very different reaction paths, opening up a plethora of choices for the generation of functional polymer networks. In the exemplary presented case ("CHic-Click"), irradiation of the copolymers with UV-A light (λirr = 365 nm) leads to cross-linking (network formation) and surface attachment simultaneously. The azide units remain intact during this cross-linking step, and alkyne-modified (bio)molecules can be bound through click reactions. Biofunctionalization of the polymer network with alkynylated streptavidin, followed by application of biotin-conjugated antibody and a model analyte, highlights the potential of these surface architectures as a toolbox which can be adapted for diverse bioanalytical applications.

Blocking-Free and Substrate-Independent Serological Microarray Immunoassays.

In bioanalytical applications, many coating strategies have been established for so-called "blocking" of the surfaces. However, most of the procedures developed so far require additional processing steps for surface blocking and small variations in the blocking efficiency result in increased background noise, which lowers the overall sensitivity of an assay. In this study, we demonstrate the preparation of a bioanalytical surface with a thin film of a photo-cross-linkable copolymer that is transformed photochemically into a surface-attached hydrogel network. The presented coating is directly applicable to various plastic substrates that are used for bioassays without the need for any prior surface modification. Such a strategy allows facile one-step immobilization of biomolecules for bioanalysis and protein-repellent properties for avoiding unspecific adsorption of analyte molecules during the assay. The protein adsorption behavior of the hydrogel-coated and blank surfaces is measured by SPR with human serum and physisorption of labeled detection antibodies. We show that the hydrogel surfaces used lower unspecific background signals and background noise and thus increase the sensitivities of the microarray immunoassays.

Geometrically enhanced sensor surfaces for the selective capture of cell-like particles in a laminar flow field.

Medical wires inserted into the blood stream of patients offer an attractive perspective to capture rare cells such as circulating tumor cells in vivo. A major challenge in such systems is to achieve an efficient interaction of the desired cells with the sensing surface and avoid those cells that simply flow by the wire without any contact while floating in a laminar flow field at some small distance to the sensor surface. We describe a new strategy to increase the interaction of cells or cell-like particles to such wire-shaped sensor surfaces both from an experimental and a theoretical point of view. For model experiments, we use cell-like particles that are flowing past the profile wire in a blood-like liquid stream. In the fluid dynamics simulations, this sensor is inserted into small capillaries. The influence of geometry and orientation of the wire with respect to the surrounding capillary onto the capture behavior is studied. Parameters, such as wire diameter, profile shape, wire torsion, and orientation of it with respect to the liquid stream, induce in some cases quite strong crossflows. These crossflows enhance the contact probability compared to a straight line wire of the same length by factors of up to about 80. A general model connecting the wire geometry with the crossflow intensity and the particle capture behavior is developed. Particle capture experiments demonstrate that the identified geometric factors can improve the capture of cell-like particles in laminar fluid flows and enhance the performance of such cell sensors.

Highly Selective Capture Surfaces on Medical Wires for Fishing Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging task. CTCs are, especially at the early stages of cancer development, extremely rare cells hidden in a vast background of regular blood cells. We describe a new strategy for the isolation of CTCs from whole blood. The key component is a medical wire coated with a multilayer assembly that allows highly specific capture of EpCAM (epithelial cell adhesion molecule) positive CTCs from blood. The assembly is generated in a layer-by-layer fashion through photochemically induced C,H insertion reactions and consists of a protective layer, which shields the contacting solution from the metal, a protein resistant layer, which prevents nonspecific interactions with proteins and a layer containing the EpCAM antibodies. In vitro experiments show that these surfaces can capture tumor cells from whole blood with enrichment factors (specifically vs nonspecifically bound cells) of up to about 3000 compared to the number of leucocytes in the blood. The purity of the isolated cells is greater than 90%. After "fishing" them from the blood, the cells, still bound to the wire, can be genetically analyzed. This demonstrates that this strategy might prove useful for next generation sequencing.

Bacillus stamsii sp. nov., a facultatively anaerobic sugar degrader that is numerically dominant in freshwater lake sediment.

A novel type of anaerobic bacteria was previously isolated from profundal lake sediment by direct dilution of the sediment in mineral agar medium containing glucose and a background lawn of Methanospirillum hungatei as a syntrophic partner. The isolated bacteria grouped with aerobic Bacillus spp. according to their 16S rRNA gene sequence, and the most closely related species is Bacillus thioparans. Fermentative growth of the novel strain with glucose was possible only in the presence of syntrophic partners, and cocultures produced acetate and methane, in some cases also lactate and traces of succinate as fermentation products. In contrast, the closely related strains Bacillus jeotgali and Bacillus sp. strain PeC11 are able to grow with glucose axenically by mixed acid fermentation yielding lactate, acetate, formate, succinate, and ethanol as fermentation products. Alternatively, the isolated strain grew anaerobically in pure culture if pyruvate was added to glucose-containing media, and lactate, acetate and formate were the major fermentation products, but the strain never produced ethanol. Aerobic growth was found with a variety of organic substrates in the presence of partly reduced sulfur compounds. In the absence of sulfide and oxygen, nitrate served as an electron acceptor. Strain BoGlc83 was characterized as the type strain of a new species for which the name Bacillus stamsii sp. nov. (DSM 19598=JCM 30025) is proposed.

Raising the shields: PCR in the presence of metallic surfaces protected by tailor-made coatings.

The implementation of PCR reactions in the presence of metallic surfaces is interesting for the generation of novel bioanalytical devices, because metals exhibit high mechanical stability, good thermal conductivity, and flexibility during deformation. However, metallic substrates are usually non-compatible with enzymatic reactions such as PCR due to poisoning of the active center of the enzyme or nonspecific adsorption of the enzymeto the metal surface, which could result in protein denaturation. We present a method for the generation of polymer coatings on metallic surfaces which are designed to minimize protein adsorption and also prevent the release of metal ions. These coatings consist of three layers covalently linked to each other; a self-assembled monolayer to promote adhesion, a photochemically generated barrier layer and a photochemically generated hydrogel. The coatings can be deposited onto aluminum, stainless steel, gold and copper surfaces. We compare PCR efficiencies in the presence of bare metallic surfaces with those of surfaces treated with the novel coating system.