Development of a Gold Nanoparticle-Based Colorimetric Sensor for Water for Injection At-Line Impurity Testing.

The best-known rapid test using gold nanoparticles (AuNPs) is the human chorionic gonadotropin pregnancy test. AuNPs are a powerful tool in point-of-care testing because of their flexibility, modifiability, and visibility. Here, we report a method to detect impurities for at-line process control in water-for-injection (WFI) manufacturing through the example of endotoxins. If a distinct concentration of these amphipathic molecules, originated from gram-negative bacteria, enters the human body, it will result in septic shock, followed by organ failure and possibly death. Every fluid given parenterally is subject to strict regulatory requirements and therefore endotoxin testing. Through use of traditional methods like the limulus amebocyte lysate (LAL) test, it takes more than 2 h to complete. With the presented method, one-fifth of the sample volume is sufficient compared with the LAL test. Once the assay components have been mixed, the result can be interpreted visually within 2 min without the use of further instruments.

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy.

Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy - a fast, real-time, and label-free bio-sensing method - represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme-linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target-monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme-linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target-layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance-based system for continuous process monitoring.

Osteogenic nanostructured titanium surfaces with antibacterial properties under conditions that mimic the dynamic situation in the oral cavity.

The study aim was to assess the impact of different surface nanofeatures on otherwise smooth titanium surfaces on bacterial adhesion as well as on their osteogenic potential. Bacterial adhesion was assessed in the presence of saliva under static and dynamic conditions to approximate both sub- and supragingival conditions in the oral cavity as the gingival seal will be affected by implantation. The ultimate goal was to develop a surface that will reduce biofilm formation but still support osseointegration in vivo. To this end nanotubular or nanopitted surfaces were created on electropolished titanium via electrochemical anodization procedures. Sandblasted/acid etched surfaces (SBAE) were used as a microrough reference. Bacterial adhesion was studied using saliva-precoated samples with S. sanguinis as a typical early colonizer of the oral cavity; osteogenic differentiation was assessed with human bone marrow stromal cells. While bacterial adhesion was reduced on all microsmooth surfaces to an average of 17% surface coverage compared to 61% on SBAE under static conditions, under dynamic conditions the nanopitted surface had a significant impact on bacterial adhesion. Here fluid flow removed all bacteria. By comparison, the reduction on the nanotubular surface was only similar to that of the SBAE reference. We hypothesise the underlying cause to be an effect of the surface morphology on the structure and composition of the saliva precoating that reduces its stability, giving rise to a self-cleaning effect. In addition, no negative influence on the osteogenic potential of the nanopitted surface could be determined by alkaline phosphatase activity, mineralization behaviour or gene expression; it remained on a par with the tissue culture plastic control. Thus, nanopitting seems to be a promising surface treatment candidate for dental implants to reduce infection related complications without compromising the implant integration.

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: Dedicated to Prof. em. Dr. Dr. H.C. Karl Schügerl on the occasion of his 90th birthday.

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.

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.

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System.

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

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.

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 Abeta, 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 in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's 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.

The use of respirometric measurements to determine the toxicity of textile dyes in aqueous solution and after oxidative decolourisation processes.

Selected results from the degradation of reactive-dye hydrolysates after UV irradiation, ozonation and sodium peroxodisulphate (NaPS) treatment are presented. Reactive dyes with representative chromophores and anchor groups were chosen for the research project. Different stages of oxidative decolourisation were examined and determined by water parameters for biological degradation (BOD). The paper focuses on toxicity tests with Pseudomonas putida to consider whether the oxidative treatments result in products with a risk for the environment. Tests were performed with the AQUALYTIC Sensomat System, which measures biological oxygen demand (BOD). It was determined that the chosen oxidative treatments had as a rule no bearing on respiration of P. putida. Experiments with hydrolysates after short-term UV irradiation resulted in a slightly increased but not long-lasting toxicity in comparison with treatments with ozone or NaPS. Toxic effects were found in tests with hydrolysates of metalliferous dyes. During oxidative treatment, metals were liberated from the chromophores. This did cause complete inhibition of respiration of P. putida. Dye Blue E, a member of a dye class with chlorotriazine anchor groups, was itself found to be toxic, caused by the reactivity of the anchor group. The hydrolysate is only of minor toxicity.