Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut

Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest kcat/KM value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane. © 2006 Elsevier Ltd. All rights reserved

Retention of copper(II) metal ions in a silicon-glass microfluidic device

This work describes the construction of a silicon microchip for retention of copper(II) metal ions. Conventional photolithographic process was applied to transfer the generated pattern to silicon wafers. Using Reactive Ion Etching (RIE), SF6 based, channels 50 mu m wide and 10 mu m deep were produced. The channels were sealed with borosilicate glass using anodic bonding process. The surface of the channels were modified with N-(beta-aminoethyl)-gamma-aminopropyl-trimetoxysilane through a silanization reaction to promote the adsorption of copper(II) ions. An amperometric detector was placed at the microchip outlet and copper(II) ions were detected by a gold electrode at 0 V (against Ag/AgCl(KCl sat.) reference electrode). Copper(II) ions were retained and eluted with HCl 50 mu mol L-1 in a micro-flow system at a flow rate about 100 mu L min(-1). Reproducibility in the peak area and height were about 4.6 % and 10 %, respectively, for three consecutive injections of 600 mu L of 10 mu mol L-1 copper(II) sample.1881531153

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material

In this work, a microfluidic free-flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 μm deep and presents an internal volume of 1.42 μL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83 mA/mm2 that corresponds to a power dissipation per unit volume of running electrolyte of 172 mW/μL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Lab-on-a-chip biosensor for glucose based on a packed immobilized enzyme reactor

In this work, the development of a packed immobilized enzyme reactor (IMER) and its integration to a capillary electrophoresis microchip is described. The present microchip design differs from others, in the fact that the same design could be used with or without the particles and, just by changing the material used to pack the IMER, different analytes can be detected. The applied procedure involves the separation of the target analyte by capillary electrophoresis (CE), which is then coupled to a post-column IMER that produces H 2O2. The H2O2 produced is finally detected downstream at the surface of a working electrode. Glucose was detected above 100 μM by packing particles modified with glucose oxidase at the end of the separation channel. The analytical performance of the microchip-CE has been demonstrated by performing the separation and detection of glucose and noradrenaline. Additions of fructose showed no effect on either the peak position or the peak magnitude of glucose. The microchip-CE-IMER was also used to quantify glucose in carbonated beverages with good agreement with other reports. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA

Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 2. Peak shape, stray capacitance, noise, and actual electronics

Although simple equivalent circuits have been used to explain the basic functioning of a capacitively coupled contactless conductivity detector ((CD)-D-4), more sophisticated models are required to take into account the effects of the spatial non-homogeneity of the solution conductivity as the electrophoretic zones pass inside the detector. The overshooting phenomenon observed in real electropherograms may be explained by modeling the coupling of the electrodes with the inner capillary with a network of resistors and capacitors and its dependence with the stray capacitance becomes evident. An even more detailed model of the cell based on electrostatics allows one to calculate the stray capacitances. For the typical geometries and materials, this capacitance is on the order of a few to hundreds of femtofarads. It was possible to demonstrate that the ground plane, sometimes used, reduces the capacitance, but does not eliminate it completely. Possible noise sources are also discussed. The electrode tightness minimizes a possible source of mechanical noise due to variation of the coupling capacitances. Thermal control should also be ensured; the calculations showed that a temperature fluctuation as low as 7 x 10(-3)degrees C induces artifacts as high as the limit of quantification of K+ in a typical electrophoretic condition, for which the technique has one of its highest sensitivities.17131207121

A toner-mediated lithographic technology for rapid prototyping of glass microchannels

A simple, fast, and inexpensive masking technology without any photolithographic step to produce glass microchannels is proposed in this work. This innovative process is based on the use of toner layers as mask for wet chemical etching. The layouts were projected in graphic software and printed on wax paper using a laser printer. The toner layer was thermally transferred from the paper to cleaned glass surfaces ( microscope slides) at 130 degrees C for 2 min. After thermal transference, the glass channel was etched using 25% ( v/v) hydrofluoric acid ( HF) solution. The toner mask was then removed by cotton soaked in acetonitrile. The etching rate was approximately 7.1 +/- 0.6 mu m min(-1). This process is economically more attractive than conventional methods because it does not require any sophisticated instrumentation and it can be implemented in any chemical/biochemical laboratory. The glass channel was thermally bonded against a flat glass cover and its analytical feasibility was investigated using capacitively coupled contactless conductivity detection ( (CD)-D-4) and laser- induced fluorescence ( LIF) detection.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.7793193

Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 1. Fundamentals

Capacitively coupled contactless conductivity detection ((CD)-D-4) is presented in a progressively detailed approach. Through different levels of theoretical and practical complexity, several aspects related to this kind of detection are addressed, which should be helpful to understand the results as well as to design a detector or plan experiments. Simulations and experimental results suggest that sensitivity depends on: 1) the electrolyte co-ion and counter-ion; 2) cell geometry and its positioning; 3) operating frequency. Undesirable stray capacitance formed due to the close placement of the electrodes is of great importance to the optimization of the operating frequency and must be minimized.17131198120

Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 2. Peak shape, stray capacitance, noise, and actual electronics

Although simple equivalent circuits have been used to explain the basic functioning of a capacitively coupled contactless conductivity detector (C 4D), more sophisticated models are required to take into account the effects of the spatial non-homogeneity of the solution conductivity as the electrophoretic zones pass inside the detector. The overshooting phenomenon observed in real electropherograms may be explained by modeling the coupling of the electrodes with the inner capillary with a network of resistors and capacitors and its dependence with the stray capacitance becomes evident. An even more detailed model of the cell based on electrostatics allows one to calculate the stray capacitances. For the typical geometries and materials, this capacitance is on the order of a few to hundreds of femtofarads. It was possible to demonstrate that the ground plane, sometimes used, reduces the capacitance, but does not eliminate it completely. Possible noise sources are also discussed. The electrode tightness minimizes a possible source of mechanical noise due to variation of the coupling capacitances. Thermal control should also be ensured; the calculations showed that a temperature fluctuation as low as 7 × 10-3°C induces artifacts as high as the limit of quantification of K+ in a typical electrophoretic condition, for which the technique has one of its highest sensitivities. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA