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Alkyd resins are oil-based polymers that have been widely used for generations in the surface coating industry and beyond. Characterization of these resins is of high importance to understand the influence of its components on its behavior, compatibility with other resins, and final quality to ensure high durability. Here, NMR spectroscopy and GPC were used for characterizing differences in the chemical structure, molecular distribution, and dispersity between oil-based and fatty acid-based alkyd polymers made from sacha inchi and linseed oils. Sancha inchi (Plukentia volubilis L.) is a fruit-bearing plant native to South America and the Caribbean, and has a rich unsaturated fatty acid content. The effect of vegetable oil and polyol selection on the synthesis of alkyd resins for coating applications was analyzed. The influence of two different synthesis methods, monoglyceride and fatty acid processes, was also compared. Important structural differences were observed using NMR: one-dimensional spectra revealed the degree of unsaturated fatty acid chains along the polyester backbone, whereas, 2D NMR experiments facilitated chemical shift assignments of all signals. GPC analysis suggested that alkyd resins with homogeneous and high molecular weights can be obtained with the fatty acid process, and that resins containing pentaerythritol may have uniform chain lengths.
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Cyanobacteria are fast-growing, genetically accessible, photoautotrophs. Therefore, they have attracted interest as sustainable production platforms. However, the lack of techniques to systematically optimize cultivation parameters in a high-throughput manner is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidic platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant, cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. This was followed by a highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt concentrations. The 1D screening results suggested that nitrate and/or phosphate may be limiting nutrients in standard cultivation media. Finally, we use 2D screening to determine the optimal N:P ratio of BG-11. Application of the improved medium composition in a high-density cultivation setup led to an increase in biomass yield of up to 15.7%. This study demonstrates that droplet-based microfluidics can decrease the volume required for cyanobacterial cultivation and screening up to a thousand times while significantly increasing the multiplexing capacity. Going forward, microfluidics have the potential to play a significant role in the industrial exploitation of cyanobacteria.
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Microfluídica , Synechocystis , Nitratos , FosfatosRESUMEN
Real-time monitoring of bioanalytes in organotypic cell cultivation devices is a major research challenge in establishing stand-alone diagnostic systems. Presently, no general technical facility is available that offers a plug-in system for bioanalytics in diversely available organotypic culture models. Therefore, each analytical device has to be tuned according to the microfluidic and interface environment of the 3D in vitro system. Herein, we report the design and function of a 3D automated culture and analysis device (3D-ACAD) which actively perfuses a custom-made 3D microbioreactor, samples the culture medium and simultaneously performs capillary-based flow ELISA. A microstructured MatriGrid® has been explored as a 3D scaffold for culturing HepaRG cells, with albumin investigated as a bioanalytical marker using flow ELISA. We investigated the effect of acetaminophen (APAP) on the albumin secretion of HepaRG cells over 96 h and compared this with the albumin secretion of 2D monolayer HepaRG cultures. Automated on-line monitoring of albumin secretion in the 3D in vitro mode revealed that the application of hepatotoxic drug-like APAP results in decreased albumin secretion. Furthermore, a higher sensitivity of the HepaRG cell culture in the automated 3D-ACAD system to APAP was observed compared to HepaRG cells cultivated as a monolayer. The results support the use of the 3D-ACAD model as a stand-alone device, working in real time and capable of analyzing the condition of the cell culture by measuring a functional analyte. Information obtained from our system is compared with conventional cell culture and plate ELISA, the results of which are presented herein.
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This work presents the application of droplet-based microfluidics for the cultivation of microspores from Brassica napus using the doubled haploid technology. Under stress conditions (e.g. heat shock) or by chemical induction a certain fraction of the microspores can be reprogrammed and androgenesis can be induced. This process is an important approach for plant breeding because desired plant properties can be anchored in the germline on a genetic level. However, the reprogramming rate of the microspores is generally very low, increasing it by specific stimulation is, therefore, both a necessary and challenging task. In order to accelerate the optimisation and development process, the application of droplet-based microfluidics can be a promising tool. Here, we used a tube-based microfluidic system for the generation and cultivation of microspores inside nL-droplets. Different factors like cell density, tube material and heat shock conditions were investigated to improve the yield of vital plant organoids. Evaluation and analysis of the stimuli response were done on an image base aided by an artificial intelligence cell detection algorithm. Droplet-based microfluidics allowed us to apply large concentration programs in small test volumes and to screen the best conditions for reprogramming cells by the histone deacetylase inhibitor trichostatin A and for enhancing the yield of vital microspores in droplets. An enhanced reprogramming rate was found under the heat shock conditions at 32 °C for about 3 to 6 days. In addition, the comparative experiment with MTP showed that droplet cultivation with lower cell density (<10 cells per droplet) or adding media after 3 or 6 days significantly positively affects the microspore growth and embryo rate inside 120 nL droplets. Finally, the developed embryos could be removed from the droplets and further grown into mature plants. Overall, we demonstrated that the droplet-based tube system is suitable for implementation in an automated, miniaturized system to achieve the induction of embryogenic development in haploid microspore stem cells of Brassica napus.
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Brassica napus , Microfluídica , Haploidia , Polen , Inteligencia Artificial , Brassica napus/genética , Células MadreRESUMEN
The alkyne, aldehyde, amine A(3)-coupling reaction, a traditional multicomponent reaction (MCR), has been investigated as a two-step flow process. The implicated aminoalkylation reaction of phenylacetylene with appropriate aldimine intermediates was catalyzed by gold nanoparticles impregnated on alumina. The aldimine formation was catalyzed by Montmorillonite K10 beforehand. The performance of the process has been investigated with respect to different reaction regimes. Usually, the A(3)-multicomponent reaction is performed as a "one-pot" process. Diversity-oriented syntheses using MCRs often have the shortcoming that only low selectivity and low yields are achieved. We have used a flow-chemistry approach to perform the A(3)-MCR in a sequential manner. In this way, the reaction performance was significantly enhanced in terms of shortened reaction time, and the desired propargylamines were obtained in high yields.
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The Eschenmoser coupling is a useful carbon-carbon bond forming reaction which has been used in various different synthesis strategies. The reaction proceeds smoothly if S-alkylated ternary thioamides or thiolactames are used. In the case of S-alkylated secondary thioamides or thiolactames, the Eschenmoser coupling needs prolonged reaction times and elevated temperatures to deliver valuable yields. We have used a flow chemistry system to promote the Eschenmoser coupling under enhanced reaction conditions in order to convert the demanding precursors such as S-alkylated secondary thioamides and thiolactames in an efficient way. Under pressurized reaction conditions at about 220 °C, the desired Eschenmoser coupling products were obtained within 70 s residence time. The reaction kinetics was investigated and 15 examples of different building block combinations are given.
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The defined formation and expansion of droplets are essential operations for droplet-based screening assays. The volumetric expansion of droplets causes a dilution of the ingredients. Dilution is required for the generation of concentration graduation which is mandatory for many different assay protocols. Here, we describe the design of a microfluidic operation unit based on a bypassed chamber and its operation modes. The different operation modes enable the defined formation of sub-µL droplets on the one hand and the expansion of low nL to sub-µL droplets by controlled coalescence on the other. In this way the chamber acts as fluidic interface between two fluidic network parts dimensioned for different droplet volumes. Hence, channel confined droplets of about 30-40 nL from the first network part were expanded to cannel confined droplets of about 500 to about 2500 nL in the second network part. Four different operation modes were realized: (a) flow rate independent droplet formation in a self-controlled way caused by the bypassed chamber design, (b) single droplet expansion mode, (c) multiple droplet expansion mode, and (d) multiple droplet coalescence mode. The last mode was used for the automated coalescence of 12 droplets of about 40 nL volume to produce a highly ordered output sequence with individual droplet volumes of about 500 nL volume. The experimental investigation confirmed a high tolerance of the developed chamber against the variation of key parameters of the dispersed-phase like salt content, pH value and fluid viscosity. The presented fluidic chamber provides a solution for the problem of bridging different droplet volumes in a fluidic network.
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Droplet-based microfluidics is a versatile tool to reveal the dose-response relationship of different effectors on the microbial proliferation. Traditional readout parameter is the temporal development of the cell density for different effector concentrations. To determine nonlinear or unconventional dose-response relationships, data with high temporal resolution and dense concentration graduation are essential. If microorganisms with slow microbial growth kinetics are investigated, a sterile and evaporation-free long-term incubation technique is required. Here, we present a modular droplet-based screening system which was developed to solve these issues. Beside relevant technical aspects of the developed modules, the procedural workflow, and exemplary dose-response data for 1D and 2D dose-response screenings are presented.
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A double-layer chemo-chip for the characterization of liquid analytes by rapid fluorimetric imaging is described. The chemo-chip consists of an array of polymeric micro-spots prepared on a glass slide. Each spot is composed of a thin indicator layer made of PVA doped with an immobilized fluorescence dye and a top layer polymer spot with different permeation properties. The analytes can be differentiated by variations in the optical response rate of the indicator dye after its application. Consequently, different cross-linker concentrations were applied using the Nano-Plotter((TM)) which formed top layers of varying permeability. The chemo-chips were tested with the aqueous solutions of two model liquids (aqueous solutions of malonic acid and phenanthroline hydrochloride). It was found that the transition time of response had changed considerably (up to a factor of about 10) depending on different local cross linking degrees. This has resulted in time-dependent fluorescent patterns of the fluorescence images of the micro-array. The response was fast and the transition times were in the range between a few seconds to 30 s.
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The nanopositioning and nanomeasuring machine was applied for the nanotopographic characterization of polymer micro spot arrays of fluorimetric chemochips. Chemochips are arrays of fluorescence dyes in a hydrogel matrix with different response behaviors of chemical components determination of chemical and physico-chemical properties of analytes by a pattern recognition approach. For the characterization and quality control of the spots, a nanometer resolution is needed over a scan range of several millimeters. This challenge could be met by use of a scanning probe sensor in connection with a laser interferometer controlled high-precision positioning and measuring device. This way, topographic scans with the resolution of atomic force microscope could be achieved over these demandingly large ranges. The technique was used in order to determine the quality of thin film micro spots made from fluorescence dye solutions on preformed polymer films and also tested for characterization of monomolecular films in the form of micro spots. The nanotopographic measurements reflect the strong influence of solvent/matrix interaction, wetting, swelling and material transport during the application of picoliter droplets in the spotting process. The measurement clarifies the reason for the formation of roughness in the nanometer range by nano-crystal formation in the upper part of polymer film and the rim formation of micro spots during solvent evaporation. The studies show the effect of application of different numbers of droplets in a dispensing series for spot formation and prove the high importance of polymer/solvent interaction for the quality of formed micro spots as well as for spot arrays of monomolecular films.
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The formation and behaviour of micelles of sodium dodecylsulfate in water byuse of a static micro mixer were studied. Trisbipyridylruthenium(II) was applied asindicator dye, 9-methylanthracene was used for fluorescence quenching. All experimentswere carried out by a micro fluid arrangement with three syringe pumps, a 2 1 two-stepstatic micro mixer (IPHT Jena) and a on-line micro fluorimetry including a luminescencediode for excitation, a blue glass filter (BG 7, Linos), two edge filters (RG 630, Linos) anda photo counting module (MP 900, Perkin Elmer). It was possible to measure thefluorescence inside the PTFE tube (inner diameter 0.5 mm) directly. A linear dependenceof fluorescence intensity from dye concentration was observed in absence of quencher andsurfactant as expected. An aggregation number of about 62 was found in the flow raterange between 300 and 800 µL/min. The fluorescence intensity increases slightly, butsignificant with increasing flow rate, if no quencher is present. In the presence of quencher,the fluorescence intensity decreases with decreasing surfactant concentration and withenhanced flow rate. The strength of the flow rate effect on the fluorescence increases withdecreasing surfactant concentration. The size of micelles was determined in micro channelsby the micro fluorimetric method in analogy to the conventional system. The micellesextract the quencher from the solution and lower, this way, the quenching effect. The sizeof micelles was estimated and it could be shown, that the flow rate has only low effect onthe aggregation number at the investigated flow rates. The effect of flow rate andsurfactant concentration on the fluorescence in the presence of quencher was interpreted asa shift in the micelle concentration due to the shear forces. It is expected, that thefluorescence intensity is lowered, if more quencher molecules are molecular disperse distributed inside the solution. Obviously, the lowered fluorescence intensity at higher flow rates suggests a reduction of the micelle density causing an increase of quencher concentration outside the micelles.