Christian Zafiu

Liquid crystal based sensors monitoring lipase activity: A new rapid and sensitive method for cytotoxicity assays

In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer’s Disease

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimers disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Traceability of fluorescent engineered nanomaterials and their fate in complex liquid waste matrices.

The number of products containing engineered nanomaterials (ENMs) has increased due to their high industrial relevance as well as their use in diverse consumer products. At the end of their life cycle ENMs might be released to the environment and therefore concerns arise regarding their environmental impact. In order to track their fate upon disposal, it is crucial to establish methods to trace ENMs in complex environmental samples and to differentiate them from naturally-occurring nanoparticles. The goal of this study was to distinctively trace ENMs by (non-invasive) detection methods. For this, fluorescent ENMs, namely quantum dots (QDs), were distinctively traced in complex aqueous matrices, and were still detectable after a period of two months using fluorescence spectroscopy. In particular, two water-dispersible QD-species, namely CdTe/CdS QDs with N-acetyl-l-cysteine as capping agent (NAC-QDs) and surfactant-stabilized CdSe/ZnS QDs (Brij(®)58-QDs), were synthesized to examine their environmental fate during disposal as well as their potential interaction with naturally-occurring substances present in landfill leachates. When QDs were spiked into a leachate from an old landfill site, alteration processes, such as sorption, aggregation, agglomeration, and interactions with dissolved organic carbon (DOC), led to modifications of the optical properties of QDs. The spectral signatures of NAC-QDs deteriorated depending on residence time and storage temperature, while Brij(®)58-QDs retained their photoluminescence fingerprints, indicating their high colloidal stability. The observed change in photoluminescence intensity was mainly caused by DOC-interaction and association with complexing agents, such as fulvic or humic acids, typically present in mature landfill leachates. For both QD-species, the results also indicated that pH of the leachate had no significant impact on their optical properties. As a result, the unique spectroscopic fingerprints of QDs, specifically surfactant-stabilized QDs, allowed distinctive tracing in complex aqueous waste matrices in order to study their long-term behavior and ultimate fate.

Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

Abstract Early diagnostics at the preclinical stage of Alzheimer’s disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies

Parkinsons disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms. The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD. Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes. The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

Liquid crystals as optical amplifiers for bacterial detection.

Interactions of bacteria with target molecules (e.g. antibiotics) or other microorganisms are of growing interest. The first barrier for targeting gram-negative bacteria is layer of a Lipopolysaccharides (LPS). Liquid crystal (LC) based sensors covered with LPS monolayers, as presented in this study, offer a simple model to study and make use of this type of interface for detection and screening. This work describes in detail the production and application of such sensors based on three different LPS that have been investigated regarding their potential to serve as sensing layer to detect bacteria. The LPS O127:B8 in combination with a LC based sensor was identified to be most useful as biomimetic sensing surface. This LPS/LC combination interacts with three different bacteria species, one gram-positive and two gram-negative species, allowing the detection of bacterial presence regardless from their viability. It could be shown that even very low bacterial cell numbers (minimum 500 cell ml(-1)) could be detected within minutes (maximum 15 min). The readout mechanism is the adsorption of bacterial entities on surface bond LPS molecules with the LC serving as an optical amplifier.

Preparation of water-soluble, PEGylated, mixed-dispersant quantum dots, with a preserved photoluminescence quantum yield

High quantum yields, colloidal stability and surfaces amenable to diverse chemical modifications are critical objectives for quantum dot (QD) synthesis. We present an approach of QD preparation developed for achieving these criteria. In addition, the nascent QDs are already water-soluble. We then partially exchanged the N-acetyl cysteine (NAC) ligands on our core–shell–shell CdTe/CdS/ZnS QDs for dithiol-poly(ethylene glycol) (dithiol-PEG). This resulted in mixed-dispersant QDs with photoluminescence properties rivaling those of QDs synthesized under high temperature conditions and modified by ligand exchange using dithiol-PEG. Optimization of the dithiol-PEG adsorption conditions not only retained efficient surface passivation by NAC as well as luminescence properties, but also resulted in sufficiently dense PEG-grafting to confer strong colloidal stability. Since particle properties such as solubility and protein resistance critically depend on the PEG conformation and density, we also evaluated the effects of various adsorption and grafting conditions on the polymer, using ATR-FTIR and TGA analysis. Leakage of cadmium ions from the core is prevented by the ZnS-shell, which is stabilized by the remnant NAC and physicochemically shielded by the PEG-layer. Furthermore, the residual NAC has carboxylic groups that can in principle still be chemically-modified with diverse functional groups. These characteristics, particularly their excellent solubility in water, render our QDs compatible for use in biomedical and environmental applications.

Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer’s Mice with Full-blown Pathology

Oligomers of the amyloid-β (Aβ) protein are suspected to be responsible for the development and progression of Alzheimer’s disease. Thus, the development of compounds that are able to eliminate already formed toxic Aβ oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aβ oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aβ pathology in old-aged transgenic Alzheimer’s Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aβ oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aβ elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aβ oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer’s disease.

Advancements of the sFIDA method for oligomer‐based diagnostics of neurodegenerative diseases

Early diagnosis of Alzheimers disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid β (Aβ) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aβ oligomers in body fluids are hampered by the low analyte concentration and presence of Aβ monomers. The surface‐based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aβ. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α‐synuclein oligomers, and significant improvement of essential assay parameters.

Corrigendum to “Supplementary material – Traceability of fluorescent engineered nanomaterials and their fate in complex liquid waste matrices” [Environ. Pollut. 214 (July 2016), 795–805]

Corrigendum to “Supplementary material e Traceability of fluorescent engineered nanomaterials and their fate in complex liquid waste matrices” [Environ. Pollut. 214 (July 2016), 795e805] Florian Part a, , Christoph Zaba , Oliver Bixner , Christian Zafiu , Stephan Hann , Eva-Kathrin Sinner b, , Marion Huber-Humer a a Department of Water-Atmosphere-Environment, Institute of Waste Management, University of Natural Resources and Life Sciences, Muthgasse 107, 1190 Vienna, Austria b Department of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria c ICS-6 Structure Biochemistry, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52425 Jülich, Germany d Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria