Active 3-D microscaffold system with fluid perfusion for culturing in vitro neuronal networks

This work demonstrated the design, fabrication, packaging, and characterization of an active microscaffold system with fluid perfusion/nutrient delivery functionalities for culturing in vitro neuronal networks from dissociated hippocampal rat pup neurons. The active microscaffold consisted of an 8 x 8 array of hollow, microfabricated, SU-8 towers (1.0 mm or 1.5 mm in height), with integrated, horizontal, SU-8 cross-members that connect adjacent towers, thus forming a 3-D grid that is conducive to branching, growth, and increased network formation of dissociated hippocampal neurons. Each microtower in the microscaffold system contained a hollow channel and multiple fluid ports for media delivery and perfusion of nutrients to the in vitro neuronal network growing within the microscaffold system. Additionally, there were two exposed Au electrodes on the outer wall of each microtower at varying heights (with insulated leads running within the microtower walls), which will later allow for integration of electrical stimulation/recording functionalities into the active microscaffold system. However, characterization of the stimulation/recording electrodes was not included in the scope of this paper. Design, fabrication, fluid packaging, and characterization of the active microscaffold system were performed. Furthermore, use of the active microscaffold system was demonstrated by culturing primary hippocampal embryonic rat pup neurons, and characterizing cell viability within the microscaffold system.     

A field test for the detection of peroxide-based explosives

A rapid and simple field test for the detection of triacetone-triperoxide (TATP) and hexamethylenetriperoxidediamine (HMTD), two explosives which find significant illegal use, has been developed. Unknown samples are first treated with a catalase solution to remove hydrogen peroxide traces, in order to provide selectivity towards peroxide-based bleaching agents which are contained in commercial laundry detergents. Subsequently, the peroxide-based explosives are decomposed via UV irradiation, thus yielding hydrogen peroxide, which is determined by the horseradish peroxidase (POD) catalysed formation of the green radical cation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS). The limits of detection for this method are 8 x 10(-6) mol dm(-3) for TATP and 8 x 10(-7) mol dm(-3) for HMTD, respectively. As an option, p-hydroxyphenylacetic acid (pHPAA) may be used as peroxidase substrate, resulting in lower limits of detection (8 x 10(-7) mol dm(-3) for TATP and HMTD). The complete method uses a mobile setup to be applied under field conditions.     

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.     

The mass detector: A new detection system for carbohydrate and lipid analyses

Summary The mass detector was investigated as an alternative to RI detection for carbohydrate and lipid analyses with high pressure liquid chromatography. The instrument was found to be more sensitive than most RI detectors and, as gradient elution could also be carried out, the resulting chromatograms showed improved resolution.     

A new device to study ex-vivo the effects of extracorporeal photochemotherapy on the immune system

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient's blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients' therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-gamma (IFN-gamma), interleukine-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4(+) and CD8(+) T lymphocytes producing IFN-gamma, IL-2 and TNF-alpha. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.     

A new ion-exchange test for detection of ketones on the microgram scale

A test for the detection of certain ketones is based on the reaction with 1-chloro-2,4-dinitrobenzene in the presence of an anion-exchange resin in the OH(-) form, to give a violet colour on the resin bead surface.     

Studies on pollutants: A new reagent for the detection of fluoride

A new solid analytical reagent is reported for the detection and semiquantitative determination of traces of fluoride. A blue (λ_max 590 nm) trypan blue dye is liberated from an insoluble zirconium-trypan blue complex by the action of fluoride in dilute acetic acid medium. The detection limit is 0.8 ppm and the range of semiquantitative determination is 0.8 to 8 ppm. The method is simple and can be conveniently used for field detection of fluoride in polluted waters.     

A new geometry for field enhancement in surface-enhanced spectroscopy

We present calculations that indicate that for well-chosen materials a system consisting of a small sphere (diameter ?400 Å) suspended above a planar surface, is a good laser intensity amplifier and an interesting candidate for surface-enhanced spectroscopy. Use of SiC and doped InSb extends the frequency range of surface-enhanced spectroscopy to the infrared (≈200 to ≈1100 cm^?1).     

A new capillary electrophoresis end-column amperometric detection system without the need for capillary/electrode alignment

A self-aligning end-column amperometric detection system for capillary electrophoresis was constructed. In this system, the electrode and capillary were exchanged easily and the capillary/electrode alignment procedure is not required. Gold, gold/mercury amalgam, copper and carbon fiber could be used as the working electrode. The principle is in the use of two disk holders with the capillary and the electrode in the center, so that by inserting the disk holders into a groove in the working electrode port, the capillary and the electrode are automatically aligned and the distance between the capillary and the electrode is assured at 0.24 mm. The relative standard deviation obtained using five different gold/mercury amalgam microdisk electrodes for determination of cysteine was 1.5% for the migration time and 3.3% for the electrophoretic peak current. The simple and convenient system was attractive for the routine analysis by capillary electrophoresis with electrochemical detection. The system was applied to the determination of promethazine hydrochloride in human serum.     

Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5-7 and pH 3-4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflammatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.     

A new spot-test for the detection of sulphites and sulphur-dioxide

A new spot-test for sulphur dioxide and sulphites is described. The sulphur dioxide is allowed to come in contact with an acid solution of ammonium vanadate which is then treated successively with solutions of ferric chloride and ammoniacal dimethylglyoxime. The ferrous ions, produced by reaction with the reduced vanadium, react with the dimethylglyoxime to give the well-known cherry-red colour.Other reagents for the detection of ferrous ions such as o-phenanthroline and dipyridyl may be substituted for dimethylglyoxime.Thiosulphate, sulphide and nitrite react as well, but can be masked by the addition of mercuric chloride in the case of the first two; by the addition of aniline hydrochloride in the case of the nitrite.The minimum amount of sulphite detectable is 4 μg.Tests are in progress with the object of applying the method to the determination of sulphur dioxide.     

Validation of a new measuring system for water turbidity field measurements

Turbidity is an essential parameter for describing water quality by direct and indirect impacts on fish, invertebrates and aquatic plants. Currently, environmental monitoring measurements are carried out with appropriate quality by accredited testing laboratories, but there is also a need for employing the third sector, i.e. citizens and voluntaries in environmental monitoring. A device called “Secchi3000” was developed as a low-cost and simple-to-operate tool so that water quality measurements can also be carried out by non-experts and citizens. The measurement using the new device is simple: The user fills the container with water and places the measurement structure in the container. The user takes a photograph with the camera on a mobile phone through a hole in the lid. The software sends the photograph to a server, which analyses the photographs automatically. Finally, the results are returned to the user’s mobile phone and stored on a database for further analysis. In this study, the measuring system for turbidity measurements in natural waters was validated. Validation included an estimation of the limit of quantification, investigations of the influence of water colour and illumination conditions on turbidity measurement values and the estimation of measurement uncertainty. A comparison of turbidity results obtained with the new device and laboratory instrument in natural water samples was carried out, and turbidity values obtained with different mobile phones were compared. According to the validation results, the new device was appropriate for the measurement of turbidity lower than 7 FNU (Formazine Nephelometric Unit). An algorithm applied for present turbidity calculations is not fully suitable for higher turbidities. For potential routine use, this is not a major problem, since most Finnish natural waters have turbidities lower than 7 FNU. For official monitoring purposes, the limit of the quantification needs to be lower than presently achieved (1.7 FNU). Although the present configuration of the Secchi3000 device is not yet fully suitable for official environmental monitoring, it will already enable the involvement of the third sector in water quality monitoring, and in this way, citizens’ observations could then serve at least as supplementary information for reporting and surveys.     

Crosslinked plastic scintillators: A new detection system for radioactivity measurement in organic and aggressive media

The measurement of radioactive solutions containing organic or aggressive media may cause stability problems in liquid and plastic scintillation (PS) techniques. In the case of PS, this can be overcome by adding a crosslinker to the polymer structure. The objectives of this study are to synthesise a suitable crosslinked plastic scintillator (C-PS) for radioactivity determination in organic and aggressive media. The results indicated that an increase in the crosslinker content reduces the detection efficiency and a more flexible crosslinker yields higher detection efficiency. For the polymer composition studied, 2,5-diphenyloxazole (PPO) is the most adequate fluorescent solute and an increase in its concentration causes little change in the detection efficiency. The inclusion of a secondary fluorescent solute 1,4-bis-2-(5-phenyloxazolyl) benzene (POPOP) improves the C-PS radiometrical characteristics. For the final composition chosen, the synthesis of the C-PS exhibits good reproducibility with elevated yield. The obtained C-PS also displays high stability in different organic (toluene, hydrotreated vegetable oil (HVO) and methanol) and aggressive media (hydrochloric acid, nitric acid and hydrogen peroxide). Finally, the C-PS exhibits high detection efficiency both in water and in aggressive media and can also be applied in organic media showing similar or even higher detection efficiency values.     

A new method for the detection of thermal oxidation in polyethylene pipes

Dumb-bell shaped specimens containing the inner wall surface of the pipe cut from 12 polyethylene pipes of different origin were subjected to constant uniaxial tensile loads at 313 K in air. The brittleness of the inner wall surface layer of the thermally oxidized pipes manifested itself in a shift of the unstable/stable necking transition to lower stresses and longer times. With the aid of optical and scanning electron microscopy, the brittleness of the inner wall surface layer of the oxidized pipes was demonstrated in distinctive surface cracks, which were the dominant feature of the drawn samples of the oxidized pipes. No such surface cracks were observed in the non-oxidized samples. The surface crack patterns were characterized in terms of crack frequency (longitudinal and transverse), width of fragments of cracked top layer (l_B), plastic strain of the fragments of the cracked top layer (ε_pl) and thickness of the cracked top layer (H). Correlations were found that can be interpreted according to basic principles of fracture mechanics. Through knowledge of, for example, ε_pl and l_B the values of the other variables can be predicted. The thickness of the cracked top layer corrected for the reduction in thickness due to plastic deformation is approximately equal to the thickness of the oxidized layer as determined by polarized microscopy. Drawing in an Instron Tensile Testing Machine at an elongation rate of 10 mm/min at 298 K also revealed the distinctive surface cracks in the oxidized samples. On the basis of these results, a new method for the detection of thermal oxidation in polyethylene pipes is proposed.     

A new detection method for purines in thin-layer chromatography

Summary The separation of some purines by thin-layer chromatography, utilizing precoated plates with Silicagel R as stationary phase and the following mobile phases: i-butanol: methylethylketone: ammonium hydroxide 25%: water (40252015) and n-propanol: ammonium hydroxide 25%: water (80155) is reported.Visualisation of the spots has been achieved by spraying with aqueous 1% uranyl acetate and irradiating with UV both at 254 and 366 nm. The visual detection of a spot was 0.01 g.     

Studies of the effects of neonatal exposure to genistein on the developing female reproductive system

Studies have shown that developmental exposure to genistein alters murine reproductive differentiation, resulting in abnormal ovarian development (multioocyte follicles) and uterine neoplasia later in life. Further, reproductive function was altered. Prolonged estrous cyclicity was observed following neonatal genistein treatment (0.5-50 mg/kg) on Days 1-5 with dose- and age-related increase in severity. Fertility, determined at 2, 4, and 6 months, showed decreased numbers of genistein-treated females (0.5 or 5 mg/kg) delivering live pups and reduced numbers of pups. At 6 months, 60% of 0.5 mg/kg and 40% of 5 mg/kg groups delivered live pups compared to 100% of controls. At 2 months, half the mice treated with 25 mg/kg of genistein and none treated with 50 mg/kg delivered live pups, although half of the latter group showed signs of pregnancy with few small implantation sites. Ovarian function was disrupted in the low genistein-dosed mice with increased numbers of corpora lutea (CLs) compared to controls and increased ovulated oocytes following exogenous gonadotropins treatment. In contrast, mice treated with high genistein doses had decreased numbers of CLs; ovulation could be restored with exogenous gonadotropins. Thus, neonatal treatment with genistein at environmentally relevant doses caused adverse consequences on ovarian development and reproductive function.     

A new GROMOS force field for hexopyranose-based carbohydrates

A new parameter set (referred to as 45A4) is developed for the explicit-solvent simulation of hexopyranose-based carbohydrates. This set is compatible with the most recent version of the GROMOS force field for proteins, nucleic acids, and lipids, and the SPC water model. The parametrization procedure relies on: (1) reassigning the atomic partial charges based on a fit to the quantum-mechanical electrostatic potential around a trisaccharide; (2) refining the torsional potential parameters associated with the rotations of the hydroxymethyl, hydroxyl, and anomeric alkoxy groups by fitting to corresponding quantum-mechanical profiles for hexopyranosides; (3) adapting the torsional potential parameters determining the ring conformation so as to stabilize the (experimentally predominant) (4)C(1) chair conformation. The other (van der Waals and nontorsional covalent) parameters and the rules for third and excluded neighbors are taken directly from the most recent version of the GROMOS force field (except for one additional exclusion). The new set is general enough to define parameters for any (unbranched) hexopyranose-based mono-, di-, oligo- or polysaccharide. In the present article, this force field is validated for a limited set of monosaccharides (alpha- and beta-D-glucose, alpha- and beta-D-galactose) and disaccharides (trehalose, maltose, and cellobiose) in solution, by comparing the results of simulations to available experimental data. More extensive validation will be the scope of a forthcoming article. (c) 2005 Wiley Periodicals, Inc. J Comput Chem 26: 1400-1412, 2005.