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.

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.

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.

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.

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.

Polyacrylamid/silver composite particles produced via microfluidic photopolymerization for single particle-based SERS microsensorics.

A micro-continuous-flow process was applied for the preparation of swellable polyacrylamide particles incorporating silver nanoparticles. These sensor particles are formed from a mixture of a colloidal solution of silver nanoparticles and monomer by a droplet-based procedure with in situ photoinitiation of polymerization and a subsequent silver enforcement in batch. The obtained polymer composite particles show a strong SERS effect. Characteristic Raman signals of aqueous solutions of adenine could be detected down to 0.1 µM by the use of single sensor particles. The chosen example demonstrates that the composite particles are suitable for quantitative microanalytical procedures with a high dynamic range (3 orders of magnitude for adenine).

A self-seeding synthesis of Ag microrods of tuned aspect ratio: ascorbic acid plays a key role.

Control of the shape and size of nanoparticles is crucial for using them as labels or as building blocks in nanotechnology. In fact, silver has so far been considered as having the widest variety of different morphologies at the nano-scale and micro-scale levels. To make progress in these criteria, in our paper we have synthesized highly reproducible silver (Ag) microrods of controlled aspect ratios through a rapid self-seeding method. The Ag nano seeds are formed via the reduction of Ag ions in hot ethylene glycol by ascorbic acid, and the subsequent growth of microrods is controlled by further deposition of Ag atoms in the presence of poly(vinylpyrrolidone). Moreover, ascorbic acid is exclusively responsible for the rod morphology, as we describe here in detail. A very low concentration of ascorbic acid forms very few Ag microrods along with a majority of Ag colloidal particles, while random overgrowth on the surfaces is observed for higher concentrations. The critical reaction condition has been found in that the aspect ratio of the Ag microrods can be systematically tuned between 4 and 90. Also, the method in which ascorbic acid is added to the reaction medium plays a key role in controlling the aspect ratio of the Ag microrods. The non-monotonic dependence of the length and the diameter of the Ag microrods has been described by an empirical equation. The function can be interpreted by means of concentration-dependent competition between the adsorption of ligands and metal deposition.

Application of micro-segmented flow for two-dimensional characterization of the combinatorial effect of zinc and copper ions on metal-tolerant Streptomyces strains.

The cultivation and growth behavior of metal-tolerant strains of Streptomyce acidiscabies E13 and Streptomyces sp. F4 were studied under droplet-based microfluidics conditions. It was shown that the technique of micro segmented flow is well suited for the investigation of dependence of bacterial growth on different concentrations of either single metal ions or combinations of them. This study confirms higher tolerance to Zn than to Cu by our test organism. The highly resolved dose-response curves reflect two transitions between the different growth behaviors, separating initial responses to Cu concentration ranges into those with (a) intense growth, (b) moderate growth, and (c) growth inhibition. For Streptomyces sp. F4, an initial stimulation was shown in the sublethal range of zinc sulfate. Two-dimensional screenings using computer-controlled fluid actuation and in situ micro flow-through fluorimetry reflected a strong growth stimulation of strain F4 by zinc sulfate in the presence of sublethal Cu concentrations. This stimulatory effect on binary mixtures may be useful in providing optimal growth conditions in bioremediation procedures.

Local Structure of ZnO micro flowers and nanoparticles obtained by micro-segmented flow synthesis.

The micro-segmented flow technique was applied for continuous synthesis of ZnO micro- and nanoparticles with short residence times of 9.4 s and 21.4 s, respectively. The obtained particles were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Small angle X-ray scattering (SAXS) and photoluminescence spectroscopy were used to determine the size and optical properties of ZnO nanoparticles. In addition, extended X-ray absorption fine structure (EXAFS) spectroscopy was employed to investigate local structural properties. The EXAFS measurements reveal a larger degree of structural disorder in the nanoparticles than the microparticles. These structural changes should be taken into consideration while evaluating the size-dependent visible emission of ZnO nanoparticles.

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.

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.

Gold-nanoparticle-catalyzed synthesis of propargylamines: the traditional A3-multicomponent reaction performed as a two-step flow process.

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.

Synergistic effects of metal nanoparticles and a phenolic uncoupler using microdroplet-based two-dimensional approach.

A droplet-based microfluidic technique for testing multiple reagent concentrations is presented. We used this experimental approach to study combined effects of gold (AuNP) and silver nanoparticles (AgNP) with the phenolic uncoupler 2,4-dinitrophenol (DNP) with respect to the growth of Escherichia coli. In order to evaluate the toxicity of binary mixtures, we first encapsulated the E. coli cells and particle mixtures inside the microdroplets using PEEK (polyetherketone) modules. Two-dimensional concentration spaces with about 500 well separated droplets were addressed. We subsequently analyzed the cell growth, the viability and the autofluorescence intensity (metabolic activity) of the bacteria with a micro-flow-through fluorometer and photometer. Dose-dependent synergistic effects were found for the binary mixture of AgNPs and DNP, which indicated a stronger interaction in the mixture than it was expected from effect summation. For the binary mixture of DNP and AuNPs in non-toxic concentrations, we found only weak synergistic effects at low DNP concentrations. Furthermore, the non-toxic tested AuNPs causes effect summation in the binary mixture with the phenolic uncoupler. In general, we demonstrated the efficiency of a droplet-based microfluidic system for fast high-throughput screenings of binary and multiple mixtures. This work also confirmed the relevance of highly resolved droplet-based assays for the miniaturization of ecotoxicological aquatic test systems.

The Eschenmoser coupling reaction under continuous-flow conditions.

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.

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.

Multiscale materials from microcontinuous-flow synthesis: ZnO and Au nanoparticle-filled uniform and homogeneous polymer microbeads.

Tri(propylene glycol) diacrylate (TPGDA) was found to be an excellent monomer for the stabilization and dispersion of inorganic nanoparticles. Uniform nano-Au/poly(TPGDA) and nano-ZnO/poly(TPGDA) composite microbeads were synthesized in situ using a designed axisymmetric capillary-based flow-focusing microfluidic device without any additional surfactant or coupling agent. Using the designed mixing-enhanced microfluidic device, homogeneous nano-inorganic/polymer composites with a high content of nanoparticles were obtained. Morphologies of the composites were characterized by SEM, TEM, surface microscopy, dark-field microscopy and internal fluorescence.

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.

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.