Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.     

Stereoselective synthesis of (+)-polyoxamic acid based on the synthesis of chiral oxazine

A concise, stereocontrolled synthesis of (+)-polyoxamic acid was achieved. Starting from trans-oxazoline as a chiral building block, the key step involves diastereoselective oxazine formation catalyzed by palladium(0).     

Monitoring FET flow control and wall adsorption of charged fluorescent dye molecules in nanochannels integrated into a multiple internal reflection infrared waveguide

Using Si as the substrate, we have fabricated multiple internal reflection infrared waveguides embedded with a parallel array of nanofluidic channels. The channel width is maintained substantially below the mid-infrared wavelength to minimize infrared scattering from the channel structure and to ensure total internal reflection at the channel bottom. A Pyrex slide is anodically bonded to the top of the waveguide to seal the nanochannels, while simultaneously enabling optical access in the visible range from the top. The Si channel bottom and sidewalls are thermally oxidized to provide an electrically insulating barrier, and the Si substrate surrounding the insulating SiO(2) layer is selectively doped to function as a gate. For fluidic field effect transistor (FET) control, a DC potential is applied to the gate to manipulate the surface charge on SiO(2) channel bottom and sidewalls and therefore their zeta-potential. Depending on the polarity and magnitude, the gate potential can accelerate, decelerate, or reverse the flow. Here, we demonstrate that this nanofluidic infrared waveguide can be used to monitor the FET flow control of charged, fluorescent dye molecules during electroosmosis by multiple internal reflection Fourier transform infrared spectroscopy. Laser scanning confocal fluorescence microscopy is simultaneously used to provide a comparison and verification of the IR analysis. Using the infrared technique, we probe the vibrational modes of dye molecules, as well as those of the solvent. The observed infrared absorbance accounts for the amount of dye molecules advancing or retracting in the nanochannels, as well as adsorbing to and desorbing from the channel bottom and sidewalls.     

Synthesis and excited-state photodynamics of a chlorin-bacteriochlorin dyad--through-space versus through-bond energy transfer in tetrapyrrole arrays.

Understanding energy transfer among hydroporphyrins is of fundamental interest and essential for a wide variety of photochemical applications. Toward this goal, a synthetic free base ethynylphenylchlorin has been coupled with a synthetic free base bromobacteriochlorin to give a phenylethyne-linked chlorin-bacteriochlorin dyad (FbC-pe-FbB). The chlorin and bacteriochlorin are each stable toward adventitious oxidation because of the presence of a geminal dimethyl group in each reduced pyrrole ring. A combination of static and transient optical spectroscopic studies indicate that excitation into the Qy band of the chlorin constituent (675 nm) of FbC-pe-FbB in toluene results in rapid energy transfer to the bacteriochlorin constituent with a rate of approximately (5 ps)(-1) and efficiency of >99%. The excited bacteriochlorin resulting from the energy-transfer process in FbC-pe-FbB has essentially the same fluorescence characteristics as an isolated monomeric reference compound, namely a narrow (12 nm fwhm) fluorescence emission band at 760 nm and a long-lived (5.4 ns) Qy excited state that exhibits a significant fluorescence quantum yield (Phif=0.19). F?rster calculations are consistent with energy transfer in FbC-pe-FbB occurring predominantly by a through-space mechanism. The energy-transfer characteristics of FbC-pe-FbB are compared with those previously obtained for analogous phenylethyne-linked dyads consisting of two porphyrins or two oxochlorins. The comparisons among the sets of dyads are facilitated by density functional theory calculations that elucidate the molecular-orbital characteristics of the energy donor and acceptor constituents. The electron-density distributions in the frontier molecular orbitals provide insights into the through-bond electronic interactions that can also contribute to the energy-transfer process in the different types of dyads.     

Analysis of sewage sludge and cover soil by neutron activation analysis

The Korean government reported that in 2005, 4395 tons/day of sewage sludge were generated from sewage disposal facilities in Korea and only 11.03% of it was reused. In addition, as a direct landfill of sewage sludge was forbidden from June 2003, research for a relevant disposal technique has been increasing. In this study, the aims were to analyze the collected sewage sludge samples and to evaluate the possibility for their reuse by a comparison of the elemental contents from a sewage sludge and a cover soil. Sludge samples were collected from a sewage disposal plant in Daejeon city and the cover soil was produced by a dilution of a sewage sludge with quicklime. Instrumental neutron activation analysis was employed to determine the elemental contents in the samples. Twenty seven elements were analyzed and their concentrations were compared.     

Novel approach to microwave-assisted extraction and micro-solid-phase extraction from soil using graphite fibers as sorbent

A single-step extraction-cleanup procedure involving microwave-assisted extraction (MAE) and micro-solid-phase extraction (micro-SPE) has been developed for the analysis of polycyclic aromatic hydrocarbons (PAHs) from soil samples. Micro-SPE is a relatively new extraction procedure that makes use of a sorbent enclosed within a sealed polypropylene membrane envelope. In the present work, for the first time, graphite fiber was used as a sorbent material for extraction. MAE-micro-SPE was used to cleanup sediment samples and to extract and preconcentrate five PAHs in sediment samples prepared as slurries with addition of water. The best extraction conditions comprised of microwave heating at 50 degrees C for a duration of 20 min, and an elution (desorption) time of 5 min using acetonitrile with sonication. Using gas chromatography (GC)-flame ionization detection (FID), the limits of detection (LODs) of the PAHs ranged between 2.2 and 3.6 ng/g. With GC-mass spectrometry (MS), LODs were between 0.0017 and 0.0057 ng/g. The linear ranges were between 0.1 and 50 or 100 microg/g for GC-FID analysis, and 1 and 500 or 1000 ng/g for GC-MS analysis. Granular activated carbon was also used for the micro-SPE device but was found to be not as efficient in the PAH extraction. The MAE-micro-SPE method was successfully used for the extraction of PAHs in river and marine sediments, demonstrating its applicability to real environmental solid matrixes.     

Extraction of lead ions by electromembrane isolation

A new microextraction approach namely electromembrane isolation (EMI) coupled with capillary electrophoresis (CE) and ultraviolet (UV) detection for determining lead (Pb) ions was developed. The EMI technique involved the use of a polypropylene hollow fiber whose channel was filled with a sodium dihydrogen phosphate/sodium tetraborate buffer solution of pH 8.1, immersed in 10 mL of an aqueous sample solution. A voltage of 300 V was applied across the hollow fiber wall for 15 min to extract Pb ions. The extract was then complexed with ethylenediamminetetracetic acid (20 mM EDTA solution, at pH 3.4) for CE analysis. Satisfactory linear dynamic ranges (0.1-10 mg L(-1)), limits of detection (0.019 mg L(-1)) and good repeatability (ranging from 4.9 to 15.6%, n=3) were obtained. EMI exhibits good linearity with a correlation coefficient of 0.9935. The optimized EMI procedure was applied to determine the concentration of Pb(2+) in various matrices, such as amniotic fluid, blood serum, lipstick and urine samples.     

The deposition of osmium carbonyl clusters onto inorganic oxide surfaces: a ToF-SIMS and IR spectroscopic study of the surface species

The interaction between the osmium clusters [Os3(CO)12], [Os3(CO)10(mu-H)2], [Os3(CO)10(mu-H)(mu-OH)], and a series containing a free functional group, viz., [Os3(CO)10(mu-H)(mu-S--EH)] (where -- =alkylwedge chain or an aromatic ring, E=COO, S or O), with SiO2, ZnO and In2O3, was examined by ToF-SIMS and IR spectroscopy. While the interaction with the silica surface is mostly via an O atom or the functional group, the interaction with the ZnO and In2O3 surfaces is more complex.