Kazuhiko Tsukagoshi

Molecular recognition of mono- and di-saccharides by phenylboronic acids in solvent extraction and as a monolayer

m- and p-Hexadecyloxyphenylboronic acids (m-1 and p-1, respectively) selectively extract saccharides; the monolayer of m-1 at the air–water interface selectively responds to these saccharides, the order of the change in π–A isotherms being similar to that of the extractability.

Microchip capillary electrophoresis using on-line chemiluminescence detection.

Chemiluminescence detection was used in capillary electrophoresis integrated on a microchip. Quartz microchips have two main channels and four reservoirs. Dansyl-lysine and -glycine were separated and detected with bis[(2-(3,6,9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate as peroxyoxalate chemiluminescent reagent. These dansyl amino acids came into contact with the chemiluminescence reagent to produce visible light at the interface between the separation channel and chemiluminescence reagent-containing reservoir. The detection limit (S/N = 3) for dansyl-lysine was 1 x 10(-5) M, which corresponded to the very small mass detection limit of ca. 0.4 fmol. However, the concentration sensitivity in the present system was approximately two orders of magnitude lower than that in the conventional capillary electrophoresis-chemiluminescence detection system. The relative standard deviations of migration time and peak height for dansyl-lysine were 4.2 and 4.5%, respectively. A channel conditioning before every run and an appropriate control of voltages were needed for the reproducible results. The present system had advantages in rapid separation time (within 40 s), small (several 10 pI) and accurate sample injection method using a cross-shaped injector, and simplification and miniaturization of the detection device.

Separation and determination of phenolic compounds by capillary electrophoresis with chemiluminescence detection.

Phenolic compounds were analyzed by means of capillary electrophoresis with chemiluminescence detection. Peroxyoxalate chemiluminescence reagent was used together with dansyl chloride as a labeling reagent. The reagent concentrations, the labeling procedures, and the performance of chemiluminescence detection cells were examined for sensitive detection of phenolic compounds. Six kinds of phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol) were determined over a range of three orders of magnitude with detection limits of the order of 10(-7) M; their detection limits were ca. 10 times as low as those obtained by an ordinary fluorescence detector. A running buffer solution containing sodium dodecylsulfate and acetonitrile provided satisfactory results in the separation of 15 kinds of phenolic compounds. The combination of capillary electrophoresis with chemiluminescence detection and column concentration procedure supported the possibility that the present system could be applied to real samples such as surface and reused waters.

Specific complexation of glucose with a diphenylmethane-3,3′-diboronic acid derivative: correlation between the absolute configuration of mono- and di-saccharides and the circular dichroic activity of the complex

For the development of receptor molecules that can precisely recognize sugar molecules, we recently synthesized bis-(6-methoxyphenyl)methane-3,3′-diboronic acid 2. This compound forms 1 :1 complexes with mono- and di-saccharides and gives circular dichroism (CD) spectra specific to each saccharide. It was shown on the basis of 1H NMR spectroscopy that the complex with D-glucose is a macrocyclic compound formed by the reaction of the two boronic acids with cis-1,2-diol and trans-4-OH, 5-CH2OH moieties. Thus, compound 2 becomes CD-active because of asymmetric immobilization of the two chromophoric benzene rings by ring closure with chiral saccharides. The association constants were in the following order: D-glucose (19000 dm3 mol–1)D-talose > D-galactose > D-mannose > D-fructose (= 0 dm3 mol–1) for monosaccharides, and D-maltose (100dm3mol–1) > D-cellobiose > D-lactose > D-saccharose (= 0 dm3mol–1) for disaccharides. In particular, compound 2 showed a very high affinity toward D-glucose. D-Glucose gave a CD spectrum with positive exciton coupling whereas L-glucose gave a CD spectrum with negative exciton coupling. D-Galactose gave a CD spectrum with negative exciton coupling, whereas all other D-mono- and D-disaccharides tested herein gave CD spectra with positive exciton coupling. The results indicate that the absolute configuration of saccharides can be conveniently predicted from the sign and the strength of the CD spectra of their complex with compound 2. This means that the CD spectroscopic method using compound 2 as a receptor probe serves as a new sensory system for sugar molecules.

Compact detection cell using optical fiber for sensitization and simplification of capillary electrophoresis-chemiluminescence detection

Abstract A new and simple chemiluminescence detection cell using optical fibers was designed for capillary electrophoresis. The cell was easily combined with capillary electrophoresis equipment and the system was operated without any tedious procedures. Luminol chemiluminescence was adapted for use with this detection cell. Hydrogen peroxide and catalysts were examined in detail with respect to their usage and concentration using the cell. The detection limit for luminol was 5.0·10−10 M (14 amol; S/N=3), which was the most sensitive result reported so far. Also, a mixture of glycine, glycylglycine and glycylglycylglycine, which was labeled with isoluminol isothiocyanate, was subjected to the present system. They were sensitively detected and were baseline separated.

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).

Migration behavior of dyestuff-containing liposomes in capillary electrophoresis with chemiluminescence detection

Abstract Various types of dyestuff-containing liposomes were prepared from eosin Y or rhodaminine B, dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylcholine–dipalmitoylphosphatidylserine mixtures, and buffer solutions (trapped solutions) differing in chemical species, buffer concentrations, and pH. The dyestuff-containing liposomes were subjected to capillary electrophoresis with chemiluminescence detection, and the effects of the constituents on liposome properties were examined from the obtained electropherogram. Two peaks were typically recorded on the electropherograms; one was due to dyestuff entrapped in the liposomes and another was due to free dyestuff in the bulk solution. The changes of retention times and ratios of the two peaks easily and rapidly offered useful information as to permeability and surface charge of the liposome membranes.

Hands-Off Preparation of Monodisperse Emulsion Droplets Using a Poly(dimethylsiloxane) Microfluidic Chip for Droplet Digital PCR

A fully autonomous method of creating highly monodispersed emulsion droplets with a low sample dead volume was realized using a degassed poly(dimethylsiloxane) (PDMS) microfluidic chip possessing a simple T-junction channel geometry with two inlet reservoirs for oil and water to be loaded and one outlet reservoir for the collection of generated droplets. Autonomous transport of oil and water phases in the channel was executed by permeation of air confined inside the outlet reservoir into the degassed PDMS. The only operation required for droplet creation was simple pipetting of oil and aqueous solutions into the inlet reservoirs. Long-lasting fluid transport in the current system enabled us to create ca. 51,000 monodispersed droplets (with a coefficient of variation of <3% for the droplet diameter) in 80 min with a maximum droplet generation rate of ca. 12 Hz using a PDMS chip that had been degassed overnight. With multiple time-course measurements, the reproducibility in the current method of droplet preparation was confirmed, with tunable droplet sizes achieved simply by changing the cross-sectional dimensions of the microchannel. Furthermore, it was verified that the resultant droplets could serve as microreactors for digital polymerase chain reactions. This hands-free technique for preparing monodispersed droplets in a very facile and inexpensive fashion is intended for, but not limited to, bioanalytical applications and is also applicable to material syntheses.

Metal ion analysis using microchip CE with chemiluminescence detection based on 1,10-phenanthroline–hydrogen peroxide reaction

We developed a microchip CE method with chemiluminescence (CL) detection using the reaction of 1,10-phenanthroline and hydrogen peroxide for separation and determination of metal ions, where the metal ions acted as catalysts for the CL reaction. The microchip consisted of two microchannels that crossed at the intersection and four reservoirs that accessed the ends of the channels. The metal ions in the sample solution migrated in the channel along with 1,10-phenanthroline included in a running solution, and then mixed with hydrogen peroxide in one of the reservoirs to emit CL. The light was detected with a photomultiplier tube located just above the reservoir. Two metal ion groups, the platinum metal group (Ru(III), Rh(III), Pd(II), Os(VIII), Ir(III), and Pt(IV)) and the fourth periodic transition metal group (Cu(II), Fe(II), Co(II), and Ni(II)) were examined using the present system. The lowest detection limit was observed for Os(VIII); Os(VIII) responded over the range of 7.5x10(-12)-1.0x10(-8 )M with the detection limit of 7.5x10(-12 )M (about 38 zmol) (S/N = 3). The mixed solution of Ru(III), Rh(III), Pd(II), Os(VIII), Ir(III), and Pt(IV) could be analyzed using this system within about 2.5 min. In addition, the system was applied to the determination of Cu(II) concentration in a city water supply.

Specific complexation of saccharides with dimeric phenylboronic acid that can be detected by circular dichroism

Abstract For the development of new receptor molecules that can recognize sugar molecules, we synthesized 2,2′-dimethoxydiphenylmethane-5,5′-diboronic acid (1) and diphenyl-3,3′-diboronic acid (2). It was shown that in the presence of 1, some mono- and disaccharides result in a CD band at 275 nm. It was also shown that 2 forms 1:1 complexes with several disaccharides and gives the characteristic exciton coupling in CD spectroscopy owing to immobilization of the two phenyl rings. The results indicate that the CD spectroscopic method using 1 or 2 as receptor molecules serves as a new sensory system for sugar molecules, and the absolute configuration of disaccharides was successfully predicted from the sign of the exciton coupling of the CD spectrum by using 2.