Archaeal and Extremophilic Bacteria from Different Archaeological Excavation Sites.

Beside natural factors, human activities are important for the development of microbiomes. Thus, local soil bacterial communities are affected by recent activities such as agriculture, mining and industry. In addition, ancient human impacts dating back centuries or millennia have changed soils and can emboss the recent bacterial communities up to now, representing a certain long-term "memory of soil". Soil samples from five different archaeological excavation places were investigated for the presence of Archaea with a Next Generation Sequencing (NGS) analysis of the DNA coding for 16S r-RNA sequences. It was found that the abundance of Archaea differs strongly between less than one and more than 40 percent of bacteria. A Principal Component Analysis (PCA) of all samples shows that the archaeological excavation places can be distinguished from each other by the archaeal component of soil bacterial communities, which presents a typical pattern for each place. Most samples are marked by the dominance of Crenarchaeota, which are presented mainly by ammonia-related types. High contents of Nanoarchaeaota have been observed in one ash deposit of a historical saline and all samples of a historical tannery area. These samples are also marked by a significant presence of Dadabacteria. The specific abundancies of special Archaea-among them ammonia-oxidizing and sulphur-related types-are due obviously to former human activities and support the concept of the "ecological memory of soil".

High-frequency Contactless Sensor for the Detection of Heparin-Induced Thrombocytopenia Antibodies via Platelet Aggregation.

Heparin-induced thrombocytopenia (HIT), a severe autoimmune disorder, occurs in patients undergoing heparin therapy. The presence of platelet-activating antibodies against platelet factor 4/Heparin in the blood confirms patients suffering from HIT. The most widely used methods for HIT diagnosis are immunoassays but the results only suit to rule out HIT as the assays provide only around 50% specificity. To confirm HIT, samples with positive results in immunoassays are retested in functional assays (>98% specificity) that track platelet-activating antibodies via platelet aggregation. However, the protocols in functional assays are either time-consuming (due to the requirement of the detection of serotonin release) or require highly trained staff for the visualization of platelets. Here, we applied a cheap and easy-to-use contactless sensor, which employs high-frequency microwaves to detect the changes in the resonant frequency caused by platelet aggregation/activation. Analysis of change in conductivity and permittivity allowed us to distinguish between HIT-like (KKO) and non-HIT-like (RTO) antibodies. KKO caused a stronger reduction of conductivity of platelet samples than RTO. Our results imply that the high-frequency contactless sensor can be a promising approach for the development of a better and easier method for the detection of HIT.

Induction of embryogenic development in haploid microspore stem cells in droplet-based microfluidics.

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.

Microtoxicology by microfluidic instrumentation: a review.

Microtoxicology is concerned with the toxic effects of small amounts of substances. This review paper discusses the application of small amounts of noxious substances for toxicological investigation in small volumes. The vigorous development of miniaturized methods in microfluidics over the last two decades involves chip-based devices, micro droplet-based procedures, and the use of micro-segmented flow for microtoxicological studies. The studies have shown that the microfluidic approach is particularly valuable for highly parallelized and combinatorial dose-response screenings. Accurate dosing and mixing of effector substances in large numbers of microcompartments supplies detailed data of dose-response functions by highly concentration-resolved assays and allows evaluation of stochastic responses in case of small separated cell ensembles and single cell experiments. The investigations demonstrate that very different biological targets can be studied using miniaturized approaches, among them bacteria, eukaryotic microorganisms, cell cultures from tissues of multicellular organisms, stem cells, and early embryonic states. Cultivation and effector exposure tests can be performed in small volumes over weeks and months, confirming that the microfluicial strategy is also applicable for slow-growing organisms. Here, the state of the art of miniaturized toxicology, particularly for studying antibiotic susceptibility, drug toxicity testing in the miniaturized system like organ-on-chip, environmental toxicology, and the characterization of combinatorial effects by two and multi-dimensional screenings, is discussed. Additionally, this review points out the practical limitations of the microtoxicology platform and discusses perspectives on future opportunities and challenges.

Extremophiles in Soil Communities of Former Copper Mining Sites of the East Harz Region (Germany) Reflected by Re-Analyzed 16S rRNA Data.

The east and southeast rim of Harz mountains (Germany) are marked by a high density of former copper mining places dating back from the late 20th century to the middle age. A set of 18 soil samples from pre- and early industrial mining places and one sample from an industrial mine dump have been selected for investigation by 16S rRNA and compared with six samples from non-mining areas. Although most of the soil samples from the old mines show pH values around 7, RNA profiling reflects many operational taxonomical units (OTUs) belonging to acidophilic genera. For some of these OTUs, similarities were found with their abundances in the comparative samples, while others show significant differences. In addition to pH-dependent bacteria, thermophilic, psychrophilic, and halophilic types were observed. Among these OTUs, several DNA sequences are related to bacteria which are reported to show the ability to metabolize special substrates. Some OTUs absent in comparative samples from limestone substrates, among them Thaumarchaeota were present in the soil group from ancient mines with pH > 7. In contrast, acidophilic types have been found in a sample from a copper slag deposit, e.g., the polymer degrading bacterium Granulicella and Acidicaldus, which is thermophilic, too. Soil samples of the group of pre-industrial mines supplied some less abundant, interesting OTUs as the polymer-degrading Povalibacter and the halophilic Lewinella and Halobacteriovorax. A particularly high number of bacteria (OTUs) which had not been detected in other samples were found at an industrial copper mine dump, among them many halophilic and psychrophilic types. In summary, the results show that soil samples from the ancient copper mining places contain soil bacterial communities that could be a promising source in the search for microorganisms with valuable metabolic capabilities.

Emerging Structural and Interfacial Features of Particulate Polymers at the Nanoscale.

Particulate polymers at the nanoscale are exceedingly promising for diversified functional applications ranging from biomedical and energy to sensing, labeling, and catalysis. Tailored structural features (i.e., size, shape, morphology, internal softness, interior cross-linking, etc.) determine polymer nanoparticles' impact on the cargo loading capacity and controlled/sustained release, possibility of endocytosis, degradability, and photostability. The designed interfacial features, however (i.e., stimuli-responsive surfaces, wrinkling, surface porosity, shell-layer swellability, layer-by-layer surface functionalization, surface charge, etc.), regulate nanoparticles' interfacial interactions, controlled assembly, movement and collision, and compatibility with the surroundings (e.g., solvent and biological environments). These features define nanoparticles' overall properties/functions on the basis of homogeneity, stability, interfacial tension, and minimization of the surface energy barrier. Lowering of the resultant outcomes is directly influenced by inhomogeneity in the structural and interfacial design through the structure-function relationship. Therefore, a key requirement is to produce well-defined polymer nanoparticles with controlled characteristics. Polymers are amorphous, flexible, and soft, and hence controlling their structural/interfacial features through the single-step process is a challenge. The microfluidics reaction strategy is very promising because of its wide range of advantages such as efficient reactant mixing and fast phase transfer. Overall, this feature article highlights the state-of-the-art synthetic features of polymer nanoparticles with perspectives on their advanced applications.

Droplet-Based Screening for the Investigation of Microbial Nonlinear Dose-Response Characteristics System, Background and Examples.

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.

Novel SERS-based process analysis for label-free segmented flow screenings.

In microfluidic segmented flow processes label-free analytical techniques like surface enhanced Raman spectroscopy (SERS) can reveal the chemical composition of the individual droplet contents. The SERS system developed in this work enables a simple connection to micro segmented flow processes through miniaturization. The concept is based on the parallelization of silver/polyacrylamide composite SERS spots on a carrier plate on which the segments are deposited. The transfer of the segments allows an easy connection to existing flow processes and provides optimal conditions for Raman measurements using miniaturized spectrometers. The preparation of the SERS polymer composite was optimized in terms of the silver content in the polymer matrix to obtain a high SERS signal. The performance and long-term stability of the polymer have been successfully demonstrated. The deamination of adenine with sodium nitrite to hypoxanthine was chosen as a case study to demonstrate the capability of the novel SERS-based process analysis. A sequence of approximately one hundred segments in combination with a gradient of the nitrite concentration (0 to 0.4 mol L-1) was generated at two temperatures. The concentration of adenine and hypoxanthine were determined by using a multivariate calibration model, since the Raman spectra of both substances are overlapping. It was shown that the conversion of adenine is increased with higher nitrite concentration and temperature. A conversion of 35% was obtained at 50 °C and a conversion of 60% at 80 °C, respectively.

Microfluidic Chamber Design for Controlled Droplet Expansion and Coalescence.

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.

Metal Nano Networks by Potential-Controlled In Situ Assembling of Gold/Silver Nanoparticles.

Invited for this month's cover picture is the group of Professors Michael Köhler at the Technische Universität Ilmenau. The cover picture shows an overlay of an image of a metal nanoparticle network (blue) and sets of non-spherical metal nanoparticles of different shapes (yellow). The particles can be used in plasmonic labelling, nanoparticle-based SERS-sensing and heterogeneous catalysis. Read the full text of their Full Paper at https://doi.org/10.1002/open.201900231.

Metal Nano Networks by Potential-Controlled In Situ Assembling of Gold/Silver Nanoparticles.

Non-spherical Au/Ag nanoparticles can be generated by chemical reduction of silver ions in the presence of preformed gold nanoparticles. The process of particle formation can be controlled by concentrations of ligands and reducing agent. The formation of ellipsoidal, nanorod- and peanut-shaped nanoparticles as well as of more complex fractal nanoassemblies can be explained by changes in particle surface state, electrochemical potential formation and particle-internal self-polarization effects. It is possible to create highly fractal nanoassemblies with sizes between the mid-nanometer and the lower micrometer range. The assemblies are marked by high optical absorption and complex nano-networks of very high surface-to-volume ratios and a granular base structure.

Preparation and Deep Characterization of Composite/Hybrid Multi-Scale and Multi-Domain Polymeric Microparticles.

Polymeric microparticles were produced following a three-step procedure involving (i) the production of an aqueous nanoemulsion of tri and monofunctional acrylate-based monomers droplets by an elongational-flow microemulsifier, (ii) the production of a nanosuspension upon the continuous-flow UV-initiated miniemulsion polymerization of the above nanoemulsion and (iii) the production of core-shell polymeric microparticles by means of a microfluidic capillaries-based double droplets generator; the core phase was composed of the above nanosuspension admixed with a water-soluble monomer and gold salt, the shell phase comprised a trifunctional monomer, diethylene glycol and a silver salt; both phases were photopolymerized on-the-fly upon droplet formation. Resulting microparticles were extensively analyzed by energy dispersive X-rays spectrometry and scanning electron microscopy to reveal the core-shell morphology, the presence of silver nanoparticles in the shell, organic nanoparticles in the core but failed to reveal the presence of the gold nanoparticles in the core presumably due to their too small size (c.a. 2.5 nm). Nevertheless, the reddish appearance of the as such prepared polymer microparticles emphasized that this three-step procedure allowed the easy elaboration of composite/hybrid multi-scale and multi-domain polymeric microparticles.

Complex oscillation modes in the Belousov-Zhabotinsky reaction by weak diffusive coupling.

We study the diffusive coupling of oscillating or excitable Belousov-Zhabotinsky reaction units arranged in a square lattice array and show that for certain sizes of the units and for certain distances between the units, complex oscillation modes of individual spots occur, which manifest themselves in multi-periodic, amplitude-modulated, and multi-mode oscillations. This experimental finding can be reproduced in simulations of the FitzHugh-Nagumo model mimicking the experimental setup, suggesting that it is a generic phenomenon in systems of coupled excitable units such as excitable cell tissues or coupled oscillators such as neurons. Further analysis let us conclude that the complex oscillation modes occur close to the transition from quiescent to coupling-induced oscillations states if this transition is taking place at weak coupling strength.

Single-Step In Situ Assembling Routes for the Shape Control of Polymer Nanoparticles.

Controlling shapes of polymer nanoparticles via single-step process is a challenge due to their amorphous chemical nature. Precise regulation of interfacial interactions, electrical charging and reaction dynamics during ongoing polymerization process provides an environment where uniform nucleation, growth and in situ assembling can be realized, and hence nanoparticles of complex shapes can be obtained. In this work, it is investigated how in situ assembling of the growing nanoparticles succeeds and specifically in different manners by using cationic, anionic, polyionic, and nonionic surface-active agents in a time-dependent blended form. Micelle of molecular surfactants leads the spheres, but long chained polyelectrolytes support in situ assembling of growing spheres to form the nonspherical polymer nanoparticles in order to minimize the surface energy of a system. Similarly, a nonionic polymer promotes the movement of growing species in solution and allows tunable aggregation-based growth which produces more complexed nanoparticles. Furthermore, the application of acid, base and salt solution also contribute specific effect where unexpected size and shape of nanoparticles can be obtained. Overall, the roles of limited polarizability, solvation power, mobility, ionic strength, pH, and microfluidics for the synthesis of various shape-controlled polymer nanoparticles are presented here.

Combination of microfluidic high-throughput production and parameter screening for efficient shaping of gold nanocubes using Dean-flow mixing.

Metal nanoparticles and their special optical properties, the so-called localized surface plasmon resonance (LSPR), facilitate many applications in various fields. Due to the strong dependency of the LSPR on particle geometry, their synthesis is a challenging and time-consuming procedure especially for non-spherical shapes. In contrast, micromixers offer new experimental approaches and therefore enable the simplification of several processes. By using a zigzag micromixer (Dean-Flow-Mixer, DFM) that induces Dean-flow secondary flow patterns, we theoretically and experimentally show the mixing efficiency. Thus, we highlight the advantages of using it in the multistep synthesis of Au nanoparticles. Based on a narrow size distribution of Au nanocubes and an increased yield in combination with higher reproducibility, we depict the need for and advantage of the DFM to control the incubation times during the growth process. We further show that, by using the DFM, easy and very fast Au nanocube edge length tuning (53 nm, 58 nm, 70 nm and 75 nm) is possible by simultaneously reducing the consumption of the materials by up to 95%. We finally demonstrate the versatile abilities by using the DFM for parameter screening on examples of different halides and accessible bromide in the growth solutions. Therefore, we highlight the optimal concentration for the different growth regimes and the influences on the Au nanoparticle morphology (spheres, cubes and rods) and their defined shaping.

Stochastically reduced communities-Microfluidic compartments as model and investigation tool for soil microorganism growth in structured spaces.

Microbial community in soil is a complex and dynamic system. Using traditional culture experiments it is difficult to model the stochastic distribution of single organisms of microbial communities in the soil pore's structure. Droplet-based micro-segmented flow technique allows the transfer of the principle of stochastic confinement of stochastically reduced communities from soil micro pores into nanoliter droplets. Microfluidics was applied for the investigation and comparison of soil samples from ancient mining areas by highly resolved concentration-dependent screenings. As results, the generation, incubation, and in situ optical characterization of nanoliter droplets of suspensions of unknown soil microbial communities allowed the identification of different response characteristics toward heavy metal exposition. The investigations proved the high potential of microfluidics for investigations of soil microbial communities. It may be in the future helpful to detect bacteria and consortia with special biosorption characteristics, which could be useful for the development of biological accumulation and detoxification strategies.

Microfluidic Assisted Synthesis of Multipurpose Polymer Nanoassembly Particles for Fluorescence, LSPR, and SERS Activities.

Potential biomedical applications such as controlled delivery with sustained drug release profile demand for multifunctional polymeric particles of precise chemical composition and with welldefined physicochemical properties. The real challenge is to obtain the reproducible and homogeneous nanoparticles in a minimum number of preparation steps. Here, single-step nanoarchitectures of soft surface layered copolymer nanoparticles with a regular tuning in the size via micro flow-through assisted synthesis are reported. Interfacial copolymerization induces the controlled compartmentalization where a hydrophobic core adopts spherical shape in order to minimize the surface energy and simultaneously shelter in the hydrophilic shelllike surface layer. Surface layer can swell in the aqueous medium and allow controlled entrapping of functional hydrophobic nanoparticles in the hydrophilic interior via electrostatic interaction which can be particularly interesting for combined fluorescence activity. Furthermore, the nanoarchitecture of size and concentration controlled polymer-metal nanoassembly particles can be implemented as an ideal surface-enhanced Raman scattering substrate for detection of the trace amounts of various analytes.

Composite Sensor Particles for Tuned SERS Sensing: Microfluidic Synthesis, Properties and Applications.

Surface-enhanced Raman scattering (SERS) is a promising platform for particle-based sensor signaling, and droplet-based microfluidic systems are particularly advantageous for control of the size and composition of micro- and nanoparticles. For controlled sensing application, a high homogeneity of the sensor particles is a key requirement, and the particles with functional properties demand for the preparation in a minimum number of synthesis steps. Frequently used coflow and flow focusing arrangements, however, produce the microparticles of only larger size. To address such concern for downscaling of particle size, which is crucial for strong sensing outcome, we have used a peculiar micro cross-flow arrangement here for generating the polymer microparticles of broad size range between 30 and 600 µm along with in situ embedded silver nanoparticles. Embedded silver acts as nuclei for additional silver enforcement via silver-catalyzed silver deposition in order to realize the composite microparticles for SERS sensing. The homogeneous size and spatial distribution of silver nanoparticles inside the matrix and enforcement over the surface together with controlled pore size provides a high and homogeneous loading of polymer composite sensor. Moreover, different parameters such as analytes concentration and particles size have been studied here for SERS sensing application of biochemical molecules (amino acids and vitamins). Overall, the platform for size-tuned droplets generation, synthesis of composite microparticles, mechanism for synchronized photopolymerization-photoreduction, tuned silver enforcement, and the impacts of different analytes on differently composed microparticles are systematically investigated in this paper.

Control of shape and size of polymer nanoparticles aggregates in a single-step microcontinuous flow process: a case of flower and spherical shapes.

Controlled aggregation of polymer nanoparticles for building anisotropic nano- and microstructures via a self-assembling bottom-up process is an important strategy. Therefore, in this work, the formation of structured poly(methyl methacrylate) (PMMA) particles with diameters between lower micrometer and submicrometer range by use of a microcontinuous flow arrangement was investigated in the presence of nonionic water-soluble polymer polyvinylpyrrolidone (PVP). The investigations show that the microreaction strategy is well applicable and allows a tuning of size and shape of nanoparticles in dependence on reactant concentrations and flow rate ratios. Larger and complex structured polymer particles have been found at lower PVP concentration, whereas more compact submicron-sized particles were formed at higher PVP concentrations. The addition of ionic surfactants modulates the generation of characteristic particle shapes. The observation of intermediate states between complex flowerlike particles and simple spheres in dependence on the applied concentration of low molecular weight surfactants supports the explanation of particle formation by a mechanism with superposition of particle growth and assembling. When mixed surfactants (PVP-SDS or PVP-CTAB) are used, the final particles shape depends on the concentration of individual concentrations of surfactants and on the competition between mobility, solvation, and micelle formations.

Micro-segmented flow and multisensor-technology for microbial activity profiling.

The combination of micro-segmented flow with miniaturized flow-through multisensor-technology has been utilized for metabolite profiling of soil bacteria. Series of sub-µl segments were generated containing soil sample slurry from historic copper mining sites and exposed to heavy metal salts of copper and nickel. Segments were examined for bacterial growth and spectral properties as well as for the effect of heavy metal-treatment after different incubation times. In order to evaluate microbial growth, extinction was recorded with 4 different spectral channels. Fluorescence was measured using a microflow-through fluorometer to detect both growth and production of fluorescent dyes or metabolites. The incidence of single segments with enhanced absorption in one of the spectral channels or enhanced fluorescence was scored to detect soil microorganisms with interesting properties for further screening. The study could show that the number of vegetated segments, the density of microorganisms in the segments after cultivation and the spectral response are different for separate soil samples and different metals. Thus, the highly parallelized and miniaturized segmented flow method is a promising tool for profiling of soil samples with regard to identifying micro-organisms with interesting profiles for secondary metabolite-production.