Biomedical subjects

Kumiko Sakai-Kato

Publications and source records attributed to Kumiko Sakai-Kato.

At least 19 recordsLinked to original sources

PubMed2006

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns.

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.

Journal Article↗

PubMed2006

Cationic amylopectin derivatives as additives for analysis of proteins in capillary electrophoresis.

Positively charged amylopectin, which is a major constituent of cationic starch, was used to modify the inner surface of fused-silica capillaries by addition to the running solution, which was subsequently employed in CE. Capillaries filled with cationic amylopectin derivatives were shown to generate a stable reversed EOF in the investigated range of pH 4-8. Among the additives studied, quaternary ammonium amylopectin derivatives with high amino and low hydroxypropyl groups showed fast electroosmotic mobility and very effectively suppressed the adsorption of proteins. The run-to-run and batch-to-batch repeatability of the procedures were satisfactory with RSDs of 0.5% and 2.4%, respectively. A basic protein, alpha-chymotrypsinogen, migrated within 6 min and the theoretical plate number of it reached 560 000 plates/m.

Amylopectin↗

PubMed2005

Creation of a P450 array toward high-throughput analysis.

The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.

Cytochrome P-450 Enzyme System↗

PubMed2005

Cationic starch derivatives as dynamic coating additives for protein analysis in capillary electrophoresis.

Positively charged starch derivatives were used to modify the inner surface of fused-silica capillaries by addition to running buffer, which were subsequently employed in capillary electrophoresis (CE). Capillaries coated with the cationic starch derivatives were shown to generate a stable, reversed electroosmotic flow (EOF) in the investigated pH range of 3-9. The presented coating procedure was fast, based on a simple rinsing protocol where the polymer created a physically adsorbed, cationic polymer layer. Among the additives studied, a quaternary ammonium starch derivative showed a fast EOF mobility and effectively suppressed the adsorption of proteins. The intra- and inter-day reproducibility of the coating referring to the EOF mobility were satisfactory with relative standard deviation (RSD) of 0.27 and 1.67%, respectively. The coating enabled separation of some protein mixtures including basic proteins within l3 min with efficiencies up to 280,000 plates/m. In addition, this cationic starch derivative possessed a good solubility (about 100mg/mL), and it does not significantly contribute to the background adsorption in the UV region of 190-400 nm.

Cations↗

PubMed2005

Monolithic bioreactor immobilizing trypsin for high-throughput analysis.

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.

Bioreactors↗

PubMed2005

Analysis of amino acids and proteins using a poly(methyl methacrylate) microfluidic system.

Plastic microchips are very promising analytical devices for the high-speed analysis of biological compounds. However, due to its hydrophobicity, their surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in a significant uncontrolled adsorption on the channel walls. This paper describes the migration of fluorescence-labeled amino acids and proteins using the poly(methyl methacrylate) microchip. A cationic starch derivative significantly decreases the adsorption of analytes on the channel walls. The migration time of the analytes was related to their molecular weight and net charge or pI of the analytes. FITC-BSA migrated within 2 min, and the theoretical plate number of the peak reached 480,000 plates/m. Furthermore, proteins with a wide range of pI values and molecular weights migrated within 1 min using the microchip.

Amino Acids↗

PubMed2004

Cationic starch derivatives as dynamic coating additives for analysis of amino acids and peptides using poly(methyl methacrylate) microfluidic devices.

Plastic microchips are very promising analytical devices because they are less fragile and are suitable for mass production. However, due to their hydrophobicity, the surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in significant uncontrolled adsorption on channel walls. This paper describes the poly(methyl methacrylate) surface treatment by dynamic coating additives that considerably decreases adsorption of analytes to channel walls. Among the additives studied, quaternary ammonium starch derivatives suppressed the adsorption of fluorescently labeled amino acids and peptides most effectively. The effect was valid over the wide pH range from 2.5 to 8.0. Using a 10 mM phosphate buffer (pH 7.0) with 3% (w/v) quaternary ammonium starch as the running buffer, Asp and Glu, respectively, migrated at 54.6 and 57.6 s with efficiencies of 380 000 and 370 000 plates/m. In addition, this cationic starch derivative was found to possess good solubility and low viscosity.

Amino Acids↗

PubMed2004

Screening of inhibitors of uridine diphosphate glucuronosyltransferase with a miniaturized on-line drug-metabolism system.

Inhibition of uridine diphosphate glucuronosyltransferase (UGT), a major drug-metabolyzing enzyme, has been studied using an on-line drug-metabolism system integrated into capillary electrophoresis. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane (TMOS)-based silica matrices within a capillary in a single step under mild conditions. This microsome-immobilized capillary column allows both the metabolism of drugs and determination of the metabolites in a single capillary simultaneously, just by injecting the substrate-coenzyme mixture onto the column. Glucuronidation of acetaminophen, a widely used pharmaceutical analgesic and antipyretic agent, was investigated using this system. The glucuronidation was inhibited by 4-nitrophenol (4NP) or probenecid that was injected onto a column along with the substrate-coenzyme mixture. On the other hand, valproate did not inhibit the metabolizing reaction. The extents of inhibition using encapsulated UGT were almost the same as those obtained using free UGT. On the other hand, this electrophoretic enzyme-inhibitor assay in microfabricated devices consumes 10(4) less sample and 10(3) less microsome per experiment compared to the conventional reaction schemes. These results demonstrate that this on-line system can circumvent laborious procedures for the isolation and determination of drug metabolites from the reaction mixtures required in the conventional schemes and can provide an attractive alternative technique for the analysis of drug interactions in the metabolic pathways.

Animals↗

PubMed2004

Silica sol-gel/organic hybrid material for protein encapsulated column of capillary electrochromatography.

A new-type of sol-gel/organic hybrid composite material using gelatin or chitosan with tetramethoxysilane was developed for the bovine serum albumin (BSA)-encapsulated monolithic column for capillary electrochromatography (CEC). The composite monolith was used to immobilize BSA in a fused-silica capillary. The addition of gelatin and chitosan to the alkoxysilane enabled the enantioseparation of Trp. A very small amount of these polymers were effective for the enantioseparation. Especially, the monolithic column prepared from chitosan with tetramethoxysilane showed a high enantioselectivity for Trp enantiomers and the value (alpha' = t2/t1, t1: fast eluted enantiomer, t2: second eluted enantiomer) reached 1.15 on CEC mode. Furthermore, the composite materials exhibited a higher stability compared to the silica sol-gel column. These results showed that the sol-gel/organic hybrid composite was useful as a monolithic matrix for the BSA-encapsulated column for CEC.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2004

Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry.

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.

Amino Acid Sequence↗

PubMed2003

An enzyme-immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group.

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)(PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate)(PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.

Adsorption↗

PubMed2003

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection.

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.

Acetonitriles↗

PubMed2003

Determination of glutamine and serine in rat cerebrospinal fluid using capillary electrochromatography with a modified photopolymerized sol-gel monolithic column.

Capillary electrochromatographic separations of amino acid mixtures were studied using two modified porous photopolymerized sol-gel monolithic columns. One was modified with dimethyloctadecylchlorosilane (DMOS), and the other was modified with DMOS, followed by chlorotrimethylsilane to end-cap residual silanol groups. Prior to separation, amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole using as a mobile phase 50 mM phosphate (pH 2.5), water, and acetonitrile in the ratio of 1:1:8. Five derivatized amino acids (Asn, Phe, Ala, Ile, and Leu) were separated within 7 min. Theoretical plate numbers varied between 58700 and 105000/m. This separation method with the end-capped monolithic column was applied to rat cerebrospinal fluid. The dominant amino acid found was Gln at a concentration of 420 microM along with small quantities of Ser (54 microM).

Animals↗

PubMed2003

Investigation of structure and enantioselectivity of BSA-encapsulated sol-gel columns prepared for capillary electrochromatography.

We have developed a protein-encapsulation technique using sol-gels for the preparation of monolithic capillary columns for capillary electrochromatography. Due to the silica-based matrix used, this hydrogel generates the appropriate electroosmotic flow. Electroosmotic mobility varied according to the gels fabricated under the different gelation conditions and using different starting materials. Using attenuated total reflectance (ATR)-FT-IR, the residual silanol groups in each hydrogels could be measured without drying procedures and it was found that electroosmotic mobility decreased with a reduction in the residual silanol groups. Enantiomeric separation of D,L-Trp was evaluated using bovine serum albumin (BSA)-encapsulated column. Preparatory conditions for BSA-encapsulated columns also influenced the retention time and enantioselectivity of D,L-Trp. The gels composed of clusters with the diameter of around 1 microm. According to ATR-FT-IR study, BSA maintained its structure after encapsulation in the gel.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2003

Creation of an on-chip enzyme reactor by encapsulating trypsin in sol-gel on a plastic microchip.

Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol-gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.

Drug Compounding↗

PubMed2003

Investigation of chromatographic performances and binding characteristics of BSA-encapsulated capillary column prepared by the sol-gel method.

We have developed a novel protein-encapsulation technique using the sol-gel method for the preparation of monolithic capillary columns for capillary electrochromatography. Bovine serum albumin (BSA) was encapsulated in tetramethoxysilane-based hydrogel and the enantioselectivity was evaluated. The present work examined the effect of various factors such as running buffer pH and concentration or organic modifier on the chromatographic performances as well as binding characteristics of BSA for D,L-tryptophan (Trp). The retention and enantioseparation dramatically changed depending on the buffer pH and concentrations. These factors influenced EOF and especially binding characteristics of BSA for L-Trp. Under the optimized conditions, the BSA-encapsulated columns revealed the adequate repeatability concerning column-to-column and run-to-run.

Animals↗

PubMed2002

On-line drug-metabolism system using microsomes encapsulated in a capillary by the sol-gel method and integrated into capillary electrophoresis.

A novel microsome-encapsulation technique using the sol-gel method was developed for the on-line drug-metabolism analytical system integrated into capillary electrophoresis. This analytical system allows both the metabolism of drugs and the determination of the metabolites in a single capillary simultaneously. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane-based silica matrices within a capillary in a single step under mild conditions. The availability of this system was evaluated using UDP-glucuronyltransferase, which is one of the most important microsomal enzymes. 4-Nitrophenol and testosterone, which were metabolized by the different isoforms of UDP-glucuronyltransferase, were used as substrates. The resultant monolithic reactor showed enzymatic activity at the same level as that of the soluble form. The following separation of the unreacted substrates and metabolites in the same capillary also showed high selectivity. Furthermore, the sample amount required for one analysis decreased more than 3 orders of magnitude from conventional reaction schemes in free solution. This on-line system could largely simplify the laborious procedures which were needed in conventional analytical schemes.

Animals↗

PubMed2002

On-line trypsin-encapsulated enzyme reactor by the sol-gel method integrated into capillary electrophoresis.

A novel trypsin-encapsulation technique using the sol-gel method was developed for the preparation of an on-line enzyme reactor integrated into capillary electrophoresis. Trypsin was encapsulated in tetramethoxysilane-based hydrogel, and its enzymatic activity was evaluated using alpha-N-benzoyl-L-arginine ethyl ester and two peptides (bradykinin and [Ter8]-bradykinin). The enzyme encapsulation was carried out in a single step under mild conditions within a capillary, and 1.5-cm gel was formed at the inlet of the capillary. The resultant monolithic reactor showed excellent enzymatic activity, which was approximately 700 times higher than that in free solution, without stopping the flow. Separation of the unreacted substrates and products in the same capillary also showed high selectivity, and sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. The encapsulated trypsin maintains its substrate specificity even in a sol-gel matrix. Furthermore, the encapsulated trypsin exhibits increased stability even after continuous use compared to that in free solution.

Electrophoresis, Capillary↗