Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH)

We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The μFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of μFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The μFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.     

In situ High-pressure NMR Observations on the Formation and Dissociation of Methane Hydrate

Gas hydrates are nonstoichionmetric ice-like solid. Three types of well-known structures, sI hydrate, sII hydrate, and sH hydrate, can be formed depending on the size of the guest molecules1. Most previous work focused on the equilibrium aspects of hydrat…     

Preparation and characterization of noble metal nanocolloids by silk fibroin in situ reduction

Silk fibroin (SF) is a natural protein from silkworm. It represents Chinas resplendent civili-zation as dress materials in the past 5000 a. To this day, the silk output in China is about 90000 tons per year, which is about 70% of the world overall output. In the past decade, it has been found that silk fibroin has special properties for being used as healthy foods, cosmetics, enzyme immobilizing materials, cell culture medium, biosensor, artificial skin, artificial muscle, perme-able membran…     

In situ Raman and UV-vis spectroscopic studies of polypyrrole and poly(pyrrole-2,6-dimethyl-β-cyclodextrin)

Polypyrrole (PPy) and poly(pyrrole-2,6-dimethyl-β-cyclodextrin) [P(Py-β-DMCD)] films prepared by potential cycling in aqueous acidic solutions on indium tin oxide (ITO)-coated glass and gold electrodes were studied by in situ UV-vis and Raman spectroscopy. Characteristic UV-vis and Raman bands were identified and their dependencies on the electrode potential have been discussed. Spectroelectrochemical results reveal differences both in the position of the spectral bands and their potential dependence for PPy and P(Py-β-DMCD) films indicating interactions between polymer chains and CDs during electropolymerization process. The films were also characterized by cyclic voltammetry and FT-IR spectroscopy.     

Electrochemical and in situ spectroelectrochemical properties of metal-free and metallophthalocyanines containing α-naphtholbenzein groups on the peripheral positions

Phthalocyanine (Pc) compounds-containing α-naphtholbenzein units have been synthesized and characterized by spectroscopic data and elemental analysis. The redox properties of the compounds were investigated by cyclic and square wave voltammetry, controlled-potential coulometry and in situ spectroelectrochemistry in DMSO and compared with the free phthalonitrile ligand. The Pc compounds displayed common metal and/or ring-based reduction and oxidation processes. However, electrochemical measurements clearly suggested that the substituents involving anthraquinone units are redox active and so have a considerable effect on the redox processes of these compounds.     

Occurrence of difluorine F2 in nature--in situ proof and quantification by NMR spectroscopy

The most reactive chemical element, F(2), has been claimed not to occur in nature. First direct evidence from in?situ NMR spectroscopy now proves that elemental F(2) indeed occurs in nature as an occlusion in "antozonite" (right in the picture), a variant of fluorite (CaF(2), left).     

“In situ” EPR studies of interaction between benzene and ZSM-5 zeolites

The effect of the state of adsorbed layers on the formation of monomeric and dimeric benzene cation-radicals (CR) on ZSM-5 zeolites has been studied. Removal of benzene is shown to be accompanied by disappearance of CR, which is reversible and takes place with preservation of the sites for their stabilization.

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- (KP) ZSM-5. , KP, .

     

In situ electrochemical evaluation of anticancer drug temozolomide and its metabolites–DNA interaction

Temozolomide (TMZ) is an antineoplastic alkylating agent with activity against serious and aggressive types of brain tumours. It has been postulated that TMZ exerts its antitumor activity via its spontaneous degradation at physiological pH. The in vitro evaluation of the interaction of TMZ and its final metabolites, 5-aminoimidazole-4-carboxamide (AIC) and methyldiazonium ion, with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues. The results obtained revealed the decrease of the dsDNA oxidation peaks with incubation time, showing that TMZ and AIC/methyldiazonium ion interact with dsDNA causing its condensation. Furthermore, the experiments of the in situ TMZ and AIC/methyldiazonium ion–dsDNA interaction using the multilayer dsDNA-electrochemical biosensor confirmed the condensation of dsDNA caused by these species and showed evidence for a specific interaction between the guanosine residues and TMZ metabolites, since free guanine oxidation peak was detected. The oxidative damage caused to DNA bases by TMZ metabolites was also detected electrochemically by monitoring the appearance of the 8-oxoguanine/2,8-dyhydroxyadenine oxidation peaks. Nondenaturing agarose gel electrophoresis of AIC/methyldiazonium ion–dsDNA samples confirmed the occurrence of dsDNA condensation and oxidative damage observed in the electrochemical results. The importance of the dsDNA-electrochemical biosensor in the in situ evaluation of TMZ–dsDNA interactions is clearly demonstrated.     

Preparation and Characterization of trans‐1,4‐Polybutadiene Nanocomposites Containing in situ Generated Silica

Abstract

trans‐1,4‐Polybutadiene (tPBD) networks crosslinked free radically with dicumyl peroxide (DCP) were reinforced by in situ silica formed in a two‐step sol–gel technique. Changing the degree of crosslinking by changing the amount of DCP, or changing the amounts of the sol–gel components [tetraethoxysilane (TEOS) and dibutyltin diacetate (DBTDA)], changed the silica generated with regard to the amount precipitated, particle size, and degree of dispersion. Stress–strain measurements in continuous extension indicated good reinforcement, even at relatively low amounts of silica. Differential scanning calorimetry (DSC) indicated decreases in heat of crystallization with increases in the amounts of silica, but thermogravimetric analysis showed initial decomposition temperatures (IDT) remained relatively constant. Suggestions are made regarding interpretation of these properties in terms of the composite morphologies.     

In situ electrochemical contact angle study of hemoglobin and hemoglobin–Fe3O4 nanocomposites

The electrochemical redox-induced contact angle changes of hemoglobin droplets in the absence and presence of tetraheptylammonium-capped Fe₃O₄ nanoparticles have been explored by using in situ electrochemical contact angle measurements. The results indicate that the electrochemical redox process may lead to some structure changes of hemoglobin (Hb), which could further induce the hydrophobic-to-hydrophilic changes of the relative droplets. Our observations demonstrate that hemoglobin could self-assemble on the surface of the functionalized Fe₃O₄ nanoparticles as Hb–Fe₃O₄ nanocomposites, which may contribute to much more significant change of the electrochemical redox-induced contact angle values than that with free nano-Fe₃O₄. These results suggest that in situ electrochemical contact angle measurements could be readily applied as a new and convenient method to detect some specific biological process. Figure Schematic drawing of the possible process and contact angle changes for the self-assembly of hemoglobin on the tetraheptylammonium-capped Fe₃O₄ nanoparticles Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Nickel-catalyzed cross-coupling of phenols and arylboronic acids through an in situ phenol activation mediated by PyBroP

A new method for the Suzuki–Miyaura cross‐coupling of phenols and arylboronic acids through in situ phenol activation mediated by PyBroP is presented. The reaction proceeds efficiently by using cost‐effective, markedly stable [NiCl₂(dppp)] (dppp=1,3‐bis(diphenylphosphino)propane) as the catalyst in only 5 mol % loading, as well as in the absence of extra ligands. The method exhibits broad applicability and high efficiency towards a wide range of both phenols and boronic acids, including activated, nonactivated, deactivated, and heteroaromatic coupling partners. In addition, various functional groups, such as ether, amino, cyano, ester, and ketone groups, are compatible with this transformation. Notably, arylboronic acids containing an unprotected NH₂ group and 2‐heterocyclic boronic acids, which are generally problematic for coupling under conventional conditions, are also viable substrates, although moderate yields were obtained for sterically hindered substrates. Consequently, the in situ cross‐coupling methodology coupled with the use of an inexpensive and stable nickel catalyst provides a rapid and efficient pathway for the assembly of biaryls and heterobiaryls with structural diversity from readily available phenol compounds.     

In‐situ Synthesis and Photoluminescence of a Europium Porphyrin Complex Incorporated in the Silica Matrix

A practicable synthesis method is explored to synthesize a europium porphyrin complex in which a water‐soluble positively charged 5,10,15,20‐tetrakis(4‐trimethylammoniophenyl)porphyrin iodide, H₂TMePPI, is immobilized into the sol‐gel silica matrix and then in‐situ metallized with the Eu³⁺ ion. The product is characterized by means of the solid UV diffusion reflection spectra, fluorescence spectra, and thermal gravimetric analysis (TG). The solid UV diffusion reflection spectra show that the number of Q bands in the product is less than that of the H₂TMePPI ligand, which is one of the important characteristics of porphyrin metallization. The fluorescence spectra of the product are different from that of the silica doped with free Eu³⁺ ions, implying the different function of Eu³⁺ ions in the product. The TG curves show that the thermal stability of the Eu(III)TMePPI entrapped into silica is higher than that of the H₂TMePPI. The effect of a heat treatment and an UV‐light irradiation on the photoluminescence properties of the composite is investigated in details. The stronger interaction between Eu(III)TMePPI and SiO₂ in the composite is responsible for the different spectra.     

In situ infrared study of formate reactivity on water–gas shift and methanol synthesis catalysts

In order to investigate the methanol synthesis reaction from CO₂/H₂, a comparative study of the reactivity of formate species on different types of catalysts and catalyst supports has been carried out. Formic acid was adsorbed on water–gas shift catalysts, Cu/ZnO/Al₂O₃ methanol synthesis catalyst and ZnO/Al₂O₃ support, Cu/ZnO/ZrO₂ and Cu/ZnO/CeO₂ methanol synthesis catalysts as well as their corresponding supports ZnO/ZrO₂ and ZnO/CeO₂. Superior reactivity and selectivity of dedicated methanol synthesis catalysts was evidenced by their behavior during the subsequent heating ramp, when these samples showed the simultaneous presence of formates and methoxy species and a higher stability of these reaction intermediates in the usual temperature range for the methanol synthesis reaction.     

Synthesis and structure of hypervalent diacetoxybromobenzene and aziridination of olefins with imino-λ3-bromane generated in situ under metal-free conditions

Ligand exchange of p-CF(3)C(6)H(4)BrF(2) with acetoxy groups using AcOH and Ac(2)O affords (diacetoxybromo)benzene in a high yield, which undergoes aziridination of alkenes in the presence of TfNH(2) and sulfamate esters in one pot under mild conditions. The aziridination with TfNH(2) proceeds stereospecifically with retention of stereochemistry of olefins at room temperature using limiting amounts of olefins under transition-metal-free conditions. The one-pot aziridination procedure using sulfamate esters can be applied to the intramolecular versions.     

A facile synthesis of isoindoline and Δ-pyrrolines from chalcone and glycine by a cascade of process involving addition,in situ decarboxylation,and cyclization

In this Letter, we report a rapid one-step entry into isoindoline and Δ¹-pyrrolines from chalcone. The key step in the synthesis is the addition of glycine to chalcone. In acidic medium the reaction presumably goes through aza-Michael addition, whereas in basic medium the reaction proceeds through condensation followed by cyclization.     

Linear oxalato- and 4,4′-dipyridyldisulfide-bridged copper(II) coordination polymer involving in situ ligand synthesis

The reaction of 1,3-bis(pyridyl-4-ylthio)propan-2-one with Cu(ClO₄)₂?·?6H₂O gave a new complex {[Cu₂(4PDS)₂(ox)(H₂O)₄](ClO₄)₂} _n (4PDS?=?4,4′-dipyridyldisulfide, ox?=?oxalate), with 4PDS and ox being created by the Cu(II)-assisted oxidation of 1,3-bis(pyridyl-4-ylthio)propan-2-one. The complex determined by X-ray crystallography is a 1D polymer, in which metallacycles formed by two 4PDS and two Cu(II) ions are further bridged through ox anions. It crystallizes in the orthorhombic system, space group Fddd, with lattice parameters a?=?15.106(2), b?=?23.667(4), c?=?27.637(3)?Å and Z?=?8.     

Synthesis and Structure of a Dinuclear Copper（Ⅱ）- ditetrazolate Involving in situ Ligand Synthesis

The hydrothermal reaction of 1,2-dicyanobenzene with NaN3 in the presence of CuCl2, 2,2′-bipyridine and H2O affords a novel dinuclear copper(II)-ditetrazolate, [Cu(bpy)- (pdtz)]2 (Hpdtz = 5,5′-1,2-phenylene-ditetrazole, bpy = 2,2′-bipyridine). The pdtz ligand is generated in situ through Sharpless 2 3 cycloaddition reaction. Its crystal structure was deter- mined by single-crystal X-ray diffraction method. The crystal crystallizes in the monoclinic system, space group P21/n with a = 8.2096(16), b = 18.580(4), c = 11.838(2) , β = 103.12(3)o, V = 1758.5(6) 3, Z = 2, Mr = 863.83, Dc = 1.631 g/cm3, F(000) = 876 and μ = 1.272 mm-1. In this novel dinuclear structure, the pdtz ligand adopts a peculiar μ2-η1,η2 coordination mode.     

In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.     

Supramolecular assembly of Keggin polyoxomolybdate and 2,2’-bipyridine generated in situ from a decarboxylation coupling reaction

An assembly of Keggin polyoxomolybdate and organic substrate（Hbipy）₃[PMo₁₂O₄₀]（1,bipy=2,2’-bipyridine） was synthesized and characterized by elemental analysis,infrared spectrum,and single-crystal X-ray analysis.The ligand bipy of compound 1 was generated by a decarboxylation coupling reaction of H₂pdc（H₂pdc=pyridine-2,6-dicarboxylic acid） in situ under hydrothermal reaction conditions and the control experiments illustrate that La（NO₃）₃ or Ce（NO₃）₃,which does not appear in the final structure,is necessary for the decarboxylation coupling reaction.Moreover,compound 1 displays strong photoluminescence property in the solid state at room temperature.     

Synthesis and characterization of poly(ɛ-caprolactone)/Fe3o4 nanocomposites by in situ polymerization

A series of magnetic nanocomposites based on poly(?-caprolactone) (PCL) and Fe₃O₄ nanoparticles were prepared using a facile in situ polymerization method. The chemical structures of the PCL/Fe₃O₄ nanocomposites were characterized by Fourier transform infrared (FTIR) spectroscopy. Results of wide-angle X-ray diffraction (WAXD) showed that the incorporation of the Fe₃O₄ nanoparticles did not affect the crystallization structure of the PCL. Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology and dispersion of the Fe₃O₄ nanoparticles within the as-synthesized nanocomposites. Results of differential scanning calorimetry (DSC) and polarizing optical microscopy (POM) showed that the crystallization temperature was raised and the spherulites size decreased by the presence of Fe₃O₄ nanoparticles in the nanocomposites due to the heterogeneous nucleation effect. The thermal stability of the PCL was depressed by incorporation of Fe₃O₄ nanoparticles from thermogravimetric analysis (TGA). The superparamagnetic behavior of the PCL/Fe₃O₄ nanocomposites was testified by the superconducting quantum interference device (SQUID) magnetometer analysis. The obtained biodegradable nanocomposites will have a great potential in magnetic resonance imaging contrast and targeted drug delivery.