Elke Boschke

Selection of a DNA aptamer against norovirus capsid protein VP1

The genetically and antigenically diverse group of noroviruses is the major cause of human viral epidemic gastroenteritis worldwide. Virus detection and control are thus crucial topics when aiming at containing and preventing the resulting large and often persisting outbreaks. Aptamers provide a promising alternative to antibodies concerning their ability to bind and thus detect and influence bio-active molecules. These small, single-stranded oligonucleotides are able to bind to a multitude of possible target molecules with high affinity. For a specific target the highest affinity aptamers are found by screening a randomized library. In this work a DNA aptamer capable of binding to the norovirus genotype II.4 capsid protein VP1 was found. The general approach is thereby not limited to norovirus capsid, but could be extended to almost any kind of biologically relevant molecule. The development of the library enrichment was further computationally analyzed in order to describe the enrichment during screening. This is the basis for a later extensive characterization of both target and aptamers that could lead to insights regarding the functional coherence of both partners. An abstract model describing this coherence could be utilized to generate a target-specific library, from which future aptamer screening runs could benefit.

A Fluorescent Glycolipid-Binding Peptide Probe Traces Cholesterol Dependent Microdomain-Derived Trafficking Pathways

Background The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Aβ, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al [1] in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. Methodology/Principal Findings In accordance with Fantinis model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB). Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol. Conclusions/Significance The current work presents the characterization and trafficking behavior of a novel sphingolipid-binding fluorescent probe, the SBD peptide. We show that SBD binding to membranes is dependent on the presence of cholesterol, sphingomyelin, and complex glycolipids. In addition, SBD targeting through the endolysosomal pathway in neurons is highly sensitive to cholesterol perturbations, making it a potentially useful tool for the analysis of sphingolipid trafficking in disease models that involve changes in cholesterol metabolism and storage.

A compact and rapid aptasensor platform based on surface plasmon resonance

Aptamers are synthetic single‐stranded oligonucleotides which bind specifically to their target. They offer several advantages over antibodies. For example, aptamers can be produced under unphysiological conditions against almost any target, including toxic or pathological substances. They are also quicker and cheaper produced than antibodies, and are easy to modify without loss of activity. Furthermore, they exhibit high stability under a width range of conditions. Consequently, they make excellent receptors for the use in biosensors. This article describes the evaluation of a novel aptasensor based on the Surface Plasmon Resonance (SPR)‐system developed by the Fraunhofer Institute for Applied Optics and Precision Engineering IOF (Jena, Germany) using a thrombin–aptamer interaction as a model system. The biotin‐tagged aptamer was attached to the sensors gold surface by means of its interaction with streptavidin. Thrombin solutions of different concentrations were pumped over this surface, and the interaction was measured under buffer flow. The binding signals for the thrombin–aptamer interaction were compared to those arising from a control random‐oligonucleotide of the same size and bearing the same modifications. Using this approach, we were able to obtain reproducible, significant and stable signals with a limit of detection of about 26 nmol/L.

New Strategies for Evaluation and Analysis of SELEX Experiments

Aptamers are an interesting alternative to antibodies in pharmaceutics and biosensorics, because they are able to bind to a multitude of possible target molecules with high affinity. Therefore the process of finding such aptamers, which is commonly a SELEX screening process, becomes crucial. The standard SELEX procedure schedules the validation of certain found aptamers via binding experiments, which is not leading to any detailed specification of the aptamer enrichment during the screening. For the purpose of advanced analysis of the accrued enrichment within the SELEX library we used sequence information gathered by next generation sequencing techniques in addition to the standard SELEX procedure. As sequence motifs are one possibility of enrichment description, the need of finding those recurring sequence motifs corresponding to substructures within the aptamers, which are characteristically fitted to specific binding sites of the target, arises. In this paper a motif search algorithm is presented, which helps to describe the aptamers enrichment in more detail. The extensive characterization of target and binding aptamers may later reveal a functional connection between these molecules, which can be modeled and used to optimize future SELEX runs in case of the generation of target-specific starting libraries.

Chip calorimetry and biomagnetic separation: Fast detection of bacterial contamination at low cell titers

We present a new chip calorimeter for fast and quantitative measurement of metabolic heat rates of microorganisms attached to magnetic beads. In biomagnetic separation (BMS) experiments, Escherichia coli K12 immobilized on nonspecifically functionalized beads has a specific heat rate of around 1 pW per cell at 37°C. Therefore, at least 2 × 104 bacteria are required to exceed the calorimetric signal resolution of 20 nW. If the samples to be analyzed have the original volume of 4 mL, bacteria at less than 104 cells mL−1 should be detectable. In practice, we achieved the detection of approximately 2 × 104 cells mL−1. The method presented here might also find some applications in the investigation of biofilms and study of biomolecular interactions.

Initial phases of microbial biofilm formation on opaque, innovative anti‐adhesive surfaces using a modular microfluidic system

Biofilms can cause numerous problems, hence it is important to understand their formation on surfaces in order to develop resistant materials and avoidance strategies. Therefore, information is required regarding adhesion processes on surfaces generally and innovative anti‐adhesive coatings in particular. Our flow cell system allows biofilms to be monitored in continuous flow conditions, without removing material for postflow imaging. The shown laminar flow ensures the maintenance of highly controlled conditions for biofilm growth. However, carried simulations of the oxygen demands of Escherichia coli cultivated as biofilms under the chosen regime indicate that conditions may become anaerobic, at least at the outlet of the flow cell, after a certain period of time. We report data on the biofouling tendencies on coatings generated with the help of direct laser interference patterning on stainless steel surfaces. Data were estimated from images acquired by fluorescence microscopy. Differences between patterned and unpatterned surfaces were not found, which is in accordance with the attachment point theory. Nevertheless, it is particularly important to elucidate in future studies the behavior of microorganisms during their attachment and the effects of variables of potentially sensitive surfaces (such as hydrophobicity, nanotopography, and charge) on their adhesion.

PetriJet Platform Technology: An Automated Platform for Culture Dish Handling and Monitoring of the Contents

Due to the size of the required equipment, automated laboratory systems are often unavailable or impractical for use in small- and mid-sized laboratories. However, recent developments in automation engineering provide endless possibilities for incorporating benchtop devices. Here, the authors describe the development of a platform technology to handle sealed culture dishes. The programming is based on the Petri net method and implemented via Codesys V3.5 pbF. The authors developed a system of three independent electrical driven axes capable of handling sealed culture dishes. The device performs two difference processes. First, it automatically obtains an image of every processed culture dish. Second, a server-based image analysis algorithm provides the user with several parameters of the cultivated sample on the culture dish. For demonstration purposes, the authors developed a continuous, systematic, nondestructive, and quantitative method for monitoring the growth of a hairy root culture. New results can be displayed with respect to the previous images. This system is highly accurate, and the results can be used to simulate the growth of biological cultures. The authors believe that the innovative features of this platform can be implemented, for example, in the food industry, clinical environments, and research laboratories.

Surface plasmon resonance based detection of human serum albumin as a marker for hepatocytes activity

Techniques for monitoring cell cultures and fermentation processes not only enable prompt feedback to variations in critical parameters (e.g., media composition and metabolites) but further improve our understanding of the processes themselves. In this context, surface plasmon resonance (SPR) spectroscopy is one of the methods of choice. This technique exploits angle shifting to follow molecular interactions in real-time. Therefore, it allows samples to be characterized without additional molecular labels and time-consuming sample preparation. The immobilization of receptors onto the chip surface is one of the most challenging requirements in SPR. Especially for measurements in crude samples, it is crucial to achieve a sufficient immobilization level and block the remaining sensitive area to prevent nonspecific binding. In this article, we present a SPR-based detection system for human serum albumin (HSA). As HSA is exclusively synthesized in the liver, it can be used to characterize the specific activity of in vitro cultivated human hepatocytes. These can be cultivated in so-called multi-organ-chips, which have been developed by groups at the TU Berlin and Fraunhofer IWS for predictive preclinical substance evaluation.

A Modular Flow Cell System for Studying Biomimetic and BioinspiredAnti-Adhesive and Antimicrobial Surfaces

ABSTRACT Biofouling causes serious problems in many kinds of technical equipment. Consequently, there is great interest in developing surfaces with anti-adhesive and antimicrobial properties. Many such surfaces exist in nature, and artificial systems that mimic or are inspired by these natural systems could potentially be valuable technical surfaces. Recent studies on the interactions of bacteria with cicada wings revealed that rather than the wings being effective at repelling bacteria, after attachment the wing surface disrupts bacterial cell walls. Inspired by these structures, SmartMembranes GmbH used electrochemical precision etching to produce porous anodized aluminum oxide (AAO) membranes with structurally well-defined surface nanopatterns having pore diameters of 200–300 nm. We investigated the surfaces of these AAO membranes to assess their potential to prevent biofouling, using Escherichia coli as a model microbe. Bacterial adhesion tests were conducted using a modular flow cell system that is designed to enable on-line observation of biofilm formation in continuous flow on opaque surfaces using a fluorescence microscope. The system was adapted to permit testing of multiple samples with various dimensions and material properties simultaneously. Bacterial adhesion tests showed that AAO surfaces with pore diameters of 300 nm exhibit 99% less biofilm growth than widely used electropolished stainless steel.

A novel protocol to prepare cell probes for the quantification of microbial adhesion and biofilm initiation on structured bioinspired surfaces using AFM for single-cell force spectroscopy

We present a novel protocol that uses single‐cell force spectroscopy to characterize the bacteria‐to‐surface interactions involved in early steps of biofilm formation. Bacteria are immobilized as a monolayer by electrostatic interactions on a polyethylenimine‐coated silica bead, and the Escherichia coli‐bead complex is then glued on a tipless cantilever. We validated our new protocol by comparing to earlier published methods using single bacteria, but in contrast to these, which carry out bacterial attachment to the bead after fixation to the cantilever, our protocol results in more reliable production of usable cell probes. Measurements of interactions of E. coli with bio‐inspired surfaces by single‐cell force spectroscopy yielded comparable detachment forces to those found with the previous methods.