Autonomous capillary systems for life science research and medical diagnostics

In autonomous capillary systems (CS) minute amounts of liquid are transported owing to capillary forces. Such CSs are appealing due to their portability, flexibility, and the exceptional physical behavior of liquids in micrometer sized microchannels, in particular, capillarity and short diffusion times. CSs have shown to be a promising technology for miniaturized immunoassays in life science research and diagnostics. Building on existing experimental demonstrations of immunoassays in CSs, a theoretical model of such immunoassays is implemented, tools and CSs for performing immunoassays are developed, key functional elements of CSs such as capillary pumps and valves are explored experimentally, and a proof-of-concept of the ultimate goal of one-step immunoassays are given in this work. For the theoretical modeling of immunoassays in CSs a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance. A method using evaporation for controlling the flow rate in CSs was developed for maximum flexibility for developing assays. The method allows to use small CSs that initially are filled by capillary forces and then provide a well defined area of the liquid-air interface from which liquid can evaporate. Temperature and humidity are continuously measured and Peltier-elements are used to adjust the temperatures in multiple areas of the CSs relative to the dew-point. Thereby flow rates in the range from ~1.2 nL s−1 to ~30 pL s−1 could be achieved in the microchannels. This method was then used for screening cells for surface receptors. CSs, that do not need any peripherals for controlling flow rates become even more appealing. We explored the filling behavior of such CSs having microchannels of various length and large capillary pumps. The capillary pumps comprise microstructures of various sizes and shapes, which are spaced to encode certain capillary pressures. The spacing and shape of the microstructures is also used to orient the filling front to obtain a reliable filling behavior and to minimize the risk of entrapping air. We show how two capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in CSs. Liquid filling CSs can hardly be stopped, but in some cases it might be beneficial to do so. In a separate chapter we explore how microstructures need to be designed to use capillary forces to stop, time, or trigger liquids. Besides well-defined flow rates in CSs accurately patterned capture antibodies (cAbs) are key for performing high-sensitive surface immunoassays in CSs. We present a method compatible with mass fabrication for patterning cAbs in dense lines of up to 8 lines per millimeter. These cAbs are used with CSs that are optimized for convenient handling, pipetting of solutions, pumping of liquids such as human serum, and visualization of signals for fluorescence immunoassays to detect c-reactive protein (CRP) with a sensitivity of 0.9 ng mL−1 (7.8 pM) from 1 uL of CRP-spiked human serum, within 11 minutes, with 4 pipetting steps, and a total volume of sample and reagents of <1.5 uL. CSs for diagnostic applications have different requirements than CSs that are used as a research tool in life sciences, where a high flexibility and performance primes over the ease of use and portability of the CSs. We give a proof-of-concept for one-step immunoassays based on CSs which we think can be the base for developing portable diagnostics for point-of-care applications. All reagents are preloaded in the CSs. A sample loaded in the CSs redissolves and reconstitutes the detection antibodies (dAbs), analyte-dAb-complexes are formed and detected downstream in the CSs. A user only needs to load a sample and measure the result using a fluorescence microscope or scanner. C-reactive protein was detected in human serum at clinical concentrations within 10 minutes and using only 2 uL of sample

Diversidad patogénica dentro de poblaciones de jopo de girasol (O. cumana)

Resúmes del XII Congreso Nacional de la Sociedad Española de FitopatologíaPeer reviewe

In vitro bioassay tools for the toxicological evaluation of dioxins and dioxin-like compounds in sediments and biota

Since the middle of the 20th century, there has been increasing concern about certain historical contaminants, which due to their potential health effects (e.g. hepatotoxicity, endocrine disruption and infertility) and properties (accumulation in e.g. sediments and tissues) pose a threat to humans and wildlife. Among these contaminants, dioxin-like compounds (DLCs) can be found, including polychlorinated dibenzo p dioxins and dibenzofurans (PCDD/Fs) as well as dioxin-like polychlorinated biphenyls (DL-PCBs). Although emissions of DLCs have been reduced considerably during the past decades, legacy pollution of sediments still constitutes a secondary pollutant source following sediment re mobilizations (e.g. floods or dredging activities). This might cause the majority of the European surface waters not to reach the aims set by the European water framework directive (WFD). It is thus not astonishing that recently, the assessment of sediment and dredged material gained more and more attention and that new concepts have been developed such as clearly defined Environmental Quality Standards (EQS) for sediments and biota. While sediment assessment originally simply based on classical, instrumental methods (e.g. high resolution gas chromatography - high resolution mass spectrometry (HRGC/HRMS)), these methods due to their lacking information on sediment toxicity today are often connected to ecotoxicological and ecological methods. However, those methods often exhibit relatively unspecific endpoints (e.g. growth inhibition) and thus do not allow for a comparison to concentrations measured via HRGC/HRMS. Because many of the toxic effects of DLCs are mediated via the cytosolic aryl hydrocarbon receptor (AhR), and the strengths of activation of the AhR constitutes a specific endpoint of many cell-based in vitro bioassays, these methods are of increasing interest to regulators and risk assessors. Biological equivalent quotients (BEQs) deduced from such assays are directly comparable to toxicity equivalents (TEQs) of HRGC/HRMS analyses. The DioRAMA project is a joint research initiative between the Institute of Environmental Research at the RWTH Aachen University and the Department G3 of the German Federal Institute of Hydrology in Koblenz. Its main goal was to establish in vitro tools for the assessment of DLCs in sediment and biota to improve current risk assessment approaches. Therefore, the present thesis investigated (1) the sensitivity of various in vitro bioassays, the suitability of certain in vitro assays to be used (2) as regulatory tools, (3) to screen the uptake of DLCs by fish and (4) to be used as prioritizing tools for sediment and soil extracts. (4) Finally, the bioavailability of certain DLCs was investigated.A literature study proved the applicability of various in vitro bioassays for the screening of DLCs in a multitude of complex samples, individual compounds and mixtures. To be capable of screening trace contaminants such as DLCs, in vitro bioassays have to be comparably sensitive like instrumental techniques. The literature data reviled that some of the in vitro bioassays approximated the sensitivity limits of chemical analytical methods (~0.1 pM dioxin) and hence suggests the potential suitability of these assays to be used as additionally regulatory tools.To verify this assumption, three in vitro bioassays (RTL-W1 EROD, H4IIE-luc and H4IIE Micro EROD assay) were investigated with regard to their possible implementation into German guidelines for the management of dredged material. Evaluations of intra- and inter-laboratory performance and predicative power of the used bioassays based on extracts of sediments, differently contaminated with DLCs. Except the high sample throughput (RTL-W1 EROD) and the high linear range (H4IIE-luc), the H4IIE Micro EROD assay showed the overall best performance among the three assays and had a similar predictive power like HRGC/HRMS analyses. The H4IIE Micro EROD assay was highly sensitive and showed a satisfying repeatability and cross-laboratory reproducibility, independent of sample complexity. Hence, the H4IIE Micro EROD assay was proven to be highly suitable for the analysis of DLCs and to be used as ptential regulatory tool in the sediment management.In a further study, the uptake of sediment-borne DLCs by common roach, a fish of high ecological relevance, was chemically and bio-chemically investigated using the same differently contaminated sediment samples (see above) as exposure media. Fish was either exposed to black worm - inoculated sediments (dietary eposure) or daily fed with uncontaminated worms. Both chemical and bio-chemical investigations of whole fish extracts predominantly revealed an uptake of sediment-borne DLCs by fish, independent of the sediment DLC contamination degree. BEQs indicated the uptake to be promoted by (1) the suspended matter concentration in the water column and (2) the additional ingestion of feed/sediment (only relevant for the sediment of highest DLC contamination).While this study proved the applicability of the Micro EROD assay for challenging sample matrices such as whole fish homogenates, another study, which chemically and biochemically investigated sediment and soil samples from the river Elbe catchment area, proved the H4IIE Micro EROD assays’ applicability to be used as high throughput screening tool for large sample sets. Samples of highest EROD-inducing potential, even though raw extracts (missing clean-up) were investigated, corresponded well to the via HRGC/HRMS detected contamination hotspots along the river. A H4IIE Micro EROD assay based limit value was deduced from the DLC concentrations of the river Elbe sediment samples and might point towards a future yes/no-decision-level in German guidelines for dredged material. A final study investigated the bioavailabilty of polycyclic aromatic hydrocarbons (PAHs). A tenax desorption experiment was meant to close the gap between instrumental and toxicological results. It was shown that the cumulative concentrations of PAHs desorbing from the four differently contaminated sediments corresponded well to (1) the initial PAH concentrations in sediments, (2) to the effects observed in fish eggs of D. rerio and (3) to a certain extend to RTL-W1 BEQs. This indicated the higher contaminated sediments to pose a potentially higher threat to the aquatic environment.The present findings might contribute to future regulatory decisions in a way that in vitro bioassays such as the Micro EROD assay could be implemented into German guidelines for dredged material to be used as an additional quality measure alongside classically used instrumental analysis and this way could significantly improve current sediment assessment strategies

Dioxin-like activity of sediments of the Elbe River and associated flood areas

The aim of the present study was the determination of cytotoxic hazard potential and dioxin-like activity of sediments from the riverbed of the Elbe River and of associated flood plains. Of peculiar interest were industrial locations with high contamination. Settled particulate matter samples from the flood areas of the Elbe River were taken after the flood disaster of 2003. The riverbed samples originate from the river head down to the estuary mouth and were sampled in the year 2008, after the flood. Freeze-dried sediments were extracted with n-hexane:acetone (1:1, v/v) by means of pressurized liquid extraction (PLE). The Neutral red assay on cytotoxicity and the EROD (7-ethoxy-resorufin-o-deethylase) assay on dioxin-like activity were then applied with the resulting extracts using the RTL-W1 permanent cell line. Within this study the main focus was set on polychlorinated biphenyles (PCBs), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzodioxins and -furanes (PCDDs/Fs), which interact with the aryl hydrocarbon receptor (AhR) and cause EROD induction. Moreover, in order to identify whether PAHs, PCDDs or PCBs cause the strongest effects in the EROD assay, a multilayer fractionation was performed. Finally, results of the bioassays were compared with available analytical data to evaluate the significance of the used in-vitro bioassays.JRC.DDG.H.5-Rural, water and ecosystem resource