Application of an integrated microchip system with capillary array electrophoresis to optimization of enzymatic reactions

In this work, a combination of complementary metal-oxide semiconductor (CMOS) microchip system with capillary array electrophoresis (CAE) is demonstrated as a system for optimizing conditions for enzymatic reaction. Dimethylacridinone (DDAO)-phosphate substrate and alkaline phosphatase conjugate were selected for the enzymatic reaction, which was applicable to the enzyme-linked immunosorbent assay (ELISA) technique. Laser-induced fluorometry with a miniature semiconductor laser was used to detect the enzymatic products. The speed of the enzymatic reaction between the DDAO-phosphate and the alkaline phosphatase conjugate was investigated as a function of reaction time. The microchip-CAE detection system could determine the pH condition and the concentration of enzyme that are suitable for rapid and low-cost analysis. This result shows the feasibility of using the microchip-CAE system for application to miniaturized screening systems.     

Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay

In this work, we report Escherichia coli O157:H7 detection using antibody-immobilized capillary reactors, enzyme-linked immunosorbent assay (ELISA), and a biochip system. ELISA selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody-immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system. The detection limits of E. coli O157:H7 using the ELISA and Cy5 label-based immunoassays were determined to be 3 and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.     

Preparation of a thermally phase-separating copolymer,poly(N-isopropylacrylamide-co-N-acryloxysuccinimide), with a controlled number of active esters per polymer chain

N-isopropylacrylamide and N-acryloxysuccinimide have been copolymerized in various mixtures of terrahydrofuran and toluene using azobisisobutyronitrile as initiator. Polymerization has been conducted for 24 h at 50°C under a slightly positive pressure of nitrogen. The copolymers were assayed for active ester content by measuring the UV absorbance (259 nm) of N-hydroxysuccinimide anion, generated by reacting the copolymers with N-isopropylamine in dimethylformamide and dissolving the resulting mixture in 0.1M HEPES buffer, pH 7.5. The molecular weight and its distribution have been estimated by gel permeation chromatography. The active ester content was found to be equivalent to the comonomer feed ratio, and the major factor controlling the molecular weight was the ratio of tetrahydrofuran to toluene. Thus, the number of active esters per polymer chain could be controlled by adjustment of the comonomer feed ratio and the ratio of tetrahydrofuran to toluene. Monomer reactivity ratios for copolymerization of N-isopropylacrylamide with N-acryloxysuccinimide were also estimated. These copolymers are useful for immobilizing binding ligands such as antibodies for subsequent thermally induced precipitation immunoassays and bioseparation processes.     

A facile synthesis of polypyrrole nanotubes using a template-mediated vapor deposition polymerization and the conversion to carbon nanotubes

Polypyrrole (PPy) nanotubes with highly uniform surface and tunable wall thickness were fabricated by one-step vapor deposition polymerization (VDP) using anodic aluminium oxide (AAO) template membranes, and transformed into carbon nanotubes through a carbonization process.     

Characterization of n-butyl acrylate centered triads in poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) by isotopic labeling and two dimensional NMR

Poly(n-butylacrylate-co-carbon monoxide-co-ethylene) (polyEBC) samples prepared from 13C-labeled monomer, n-butyl acrylate, were characterized using two dimensional (2D) pulsed field gradient (PFG) 750 MHz NMR spectroscopy. To elucidate the complex structure of the terpolymer, 2D-1H/13C-heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were conducted by selectively exciting the enhanced resonances in the spectra of two polymer samples, one polymer resulting from synthesis with 1-13C-n-butylacrylate monomer and a second polymer obtained from a synthesis with 2-13C-n-butylacrylate monomer. High-resolution 2D-NMR combined with 13C-labeling of the polymer greatly simplifies the 2D-NMR spectra, selectively enhances the weak peaks from low occurrence B-centered triad structures, and aids in their resonance assignments. In all experiments, the sample temperature was 120 degrees C, to ensure a homogeneous solution and sufficient molecular mobility. Electronic Supplementary Material: Supplementary material (1D 13C NMR spectra of the 13C-labeled and unlabeled polymers) is available in the online version of this article at http://dx.doi.org/100.1007/s00216-003-2402-3.     

Photochemical reactions of a dimethacrylate compound containing a chalcone moiety in the main chain

We synthesized the new photosensitive oligomer containing a chalcone moiety in the main chain by end-capping reaction of diepoxide compound with methacrylic acid. The chalcone-epoxy oligomeric compound was synthesized with 4,4^′-dihydroxychalcone and epichlorohydrin. Investigation of the photosensitivity of the newly synthesized chalcone oligomer was carried out by using UV-Vis absorption and infrared spectroscopies under UV exposure. We observed the photodimerization behavior under UV irradiation. At the same time, we could also observe the photopolymerization of the compound with a trace amount of dimethoxyphenyl acetophenone. Thermal properties of UV-cured dimethacrylate compounds were also studied.     

Synthesis of 4-acetylisocoumarin: first total syntheses of AGI-7 and sescandelin

A practical synthetic route to 4-acetylisocoumarins and the first total synthesis of AGI-7 (5) and sescandelin (4) are described. The readily available homophthalate 8 was transformed to the vinylogous amide ester 13 in high overall yield. Upon treatment of 13 with refluxing aqueous formic acid, the desired 4-acetylisocoumarin (5) and its regioisomer 3-methyl-4-formylisocoumarin (17) were produced in a 3:1 ratio. After separation of the desired product (5) from the unwanted minor isomer, the enantioselective reduction of AGI-7 by borane in the presence of Corey's (S)-oxazaborolidine reagent afforded (+)-sescandelin (4) with a 93% ee.     

Study of hydantoin. III . Mass spectrometric behaviour in the electron impact of 2-imino-4-thiazolidinone derivatives

The mass spectral decomposition modes of a series of 2-imino-4-thiazolidinone derivatives are reported and discussed. The fragmentation patterns and mechanisms postulated have been confirmed by the analysis of the MIKE spectra and using DADI technique and the effects of heteroatoms and substituents on the MIKE spectrum of these compounds are compared with that of 3-substituted hydantoins.     

Infrared-visible and visible-visible double resonance spectroscopy of 1-hydroxy-9,10-anthraquinone-(H2O)n (n=1,2) complexes

The structures of hydrated 1-hydroxyanthraquinone complexes (1-HAQ), 1-HAQ(H2O)n=1,2, with intramolecular and intermolecular hydrogen bonding interactions were studied using laser spectroscopic methods such as laser induced fluorescence, fluorescence-detected infrared, infrared-visible hole burning, and visible-visible hole burning spectroscopy. In the 1:1 complex 1-HAQ(H2O)1, the water binds to the free carbonyl group of 1-HAQ not associated with intramolecular hydrogen bond. The second water in the 1:2 complex, 1-HAQ(H2O)2, binds to the first water of the 1:1 complex rather than other hydrogen bonding sites of 1-HAQ. A pair of two geometric isomers was produced in a supersonic jet for each of the 1:1 and 1:2 complexes. Both isomers of each complex have the same vibrational spectra in the region of the OH stretching vibration of water, but have different energies for the 0-0 band of vibronic transition due to the asymmetry of the two phenyl rings in 1-HAQ. The 0-0 bands for all four species of 1-HAQ(H2O)n=1,2 were unambiguously assigned by comparing with the results of ab initio calculations, which yielded the structures, vibrational frequencies, and relative energies of the frontier molecular orbitals.