This review focuses on biological and biomedical mass spectrometry, and covers a selection of publications in this area included in the MEDLINE database for the period 1987 - 2001. Over the last 15 years, biological and biomedical mass spectrometry has progressed out of all recognition. The development of soft ionization methods, such as electrospray ionization and matrix-assisted laser desorption ionization, has mainly contributed to the remarkable progress, because they can easily produce gas-phase ions of large, polar, and thermally labile biomolecules, such as proteins, peptides, nucleic acids and others. The innovations of ionization methods have led to remarkable progress in mass spectrometric technology and in biochemistry, biotechnology and molecular biology research. In addition, mass spectrometry is one of the powerful and effective technologies for drug discovery and development. It is applicable to studies on structural determination, drug metabolism, including pharmacokinetics and toxicokinetics, and de novo drug discovery by applying post-genomic approarches. In the present review, the innovative soft ionization methods are first discussed along with their features. Also, the characteristics of the mass spectrometers which are active in the biological and biomedical research fields are also described. In addition, examples of the applications of biological and biomedical mass spectrometry are provided.

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This review focuses on the integration of chemical and biochemical analysis systems into glass microchips for general use. By combining multiphase laminar flow driven by pressure and micro unit operations, such as mixing, reaction, extraction and separation, continuous-flow chemical processing systems can be realized in the microchip format, while the application of electrophoresis-based chip technology is limited. The performances of several analysis systems were greatly improved by microchip integration because of some characteristics of microspace, i.e., a large specific interface area, a short molecular diffusion time, a small heat capacity and so on. By applying these concepts, several different analysis systems, i.e., wet analysis of cobalt ion, multi-ion sensor, immunoassay, and cellular analysis, were successfully integrated on a microchip. These microchip technologies are promising for meeting the future demands of high-throughput chemical processing.

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M-DNA is a complex between divalent metal ions such as Zn²⁺ and duplex DNA which forms at pH 8.5. Unlike B-DNA, M-DNA does not bind ethidium so that M-DNA formation can be monitored conveniently by an ethidium fluorescence assay. M-DNA was shown to be a better conductor than B-DNA by fluorometric measurements of electron transport in donor-acceptor labelled duplexes; by direct conductivity measurements of M-DNA bound between gold electrodes and by cyclic voltammetric studies on ferrocene labelled duplexes attached to gold microelectrodes. As is the case with B-DNA, M-DNA can self-assemble into a variety of structures and is anticipated to find widespread use in nanoelectronics and biosensing.

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The dielectrophoretic (DEP) behavior of individual yeast cells (5 - 7 μm in diameter) in aqueous media was observed in a fabricated planar quadrupole microelectrode with a working area of 100 μm in diameter by an optical microscope. The yeast cells migrated in the radial direction in the working area. The DEP velocity of the cells increased as they approached the electrode. The DEP trajectory of the cells was analyzed with a theoretical equation derived previously, and the dielectrophoretic mobility was determined. The dielectrophoretic mobility was found to be affected by the viability of cells, the conductivity of the medium, and the binding of lectin protein (concanavalin A) to the cell surface. These DEP behaviors were analyzed based on the permittivities and conductivities of the cell interior and wall, and those of the medium.

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A simultaneous measurement technique for determining the migration velocity of a micrometer-sized particle in a capillary and the adsorption force to the inner surface of the capillary has been proposed. This technique is based on an electromagnetophoretic force being exerted on a micro-particle in an electrolyte solution, which is governed mainly by the electromagnetic buoyancy, when a homogeneous magnetic field is applied at a right angle to the electric current through the medium. By the electromagnetic buoyancy, micro-particles such as polystyrene, carbon and yeast were migrated perpendicular to the direction of the electric current and reached a fused-silica wall. A switching of the current direction could desorb the particle from the wall, and allowed to calculate the detaching force from the desorbing current. The migration velocity normalized to the size in the magnetic field of 10 T was increased in the order of yeast, carbon and polystyrene, while reflecting the decreasing order of the apparent conductivity of the particles. The desorption force could be measured up to 1 nN with a sensitivity of pN. The observed interaction forces of polystyrene and carbon were in the range of 250 - 600 pN with large deviations.

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Highly stereoselective, uniformly sized molecularly imprinted polymers (MIPs) for cinchona alkaloids, cinchonine (CN) and cinchonidine (CD), were prepared using methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EDMA) as a cross-linker. The MIPs were evaluated using a mixture of phosphate buffer and acetonitrile as the mobile phase. The CN- and CD-imprinted MAA-co-EDMA polymers can recognize the respective template molecule more than the other diastereomer, and afford an excellent diastereomer separation of CN and CD. In addition, the MIPs gave diastereomer separations of structurally related compounds, quinidine and quinine. The retentive and stereoselective properties of those compounds on the MIPs suggest that electrostatic and hydrophobic interactions can work to recognize these compounds. Furthermore, thermodynamic studies reveal that the entropy-driven effect is significant at mobile-phase pH 5.4, while the enthalpy-driven interactions seem to be dominant at mobile-phase pH 9.6.

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The nano-kinetics of colloidal particles and living cells with the colloidal particles were visualized by a newly developed video scope. The system of the new video scope has a feature of fine controlling the illumination conditions by using a single optical fiber. This characteristic enables one to obtain clear images of living cells and the motions of colloidal particles by light-scattering effects. In the experiments, RBL-2H3 cells and gold colloidal particles were observed. Scattering images with high contrast and a dark background like in dark-field observations could be attained. In the experiments, a pulsed laser was also applied. The results obtained in this study could validate the effectiveness and possibility of a new video scope for applications to biological and biomedical fields.

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MALDI-TOF mass spectrometry was used to detect intracellular molecules from a single intact cell on monolayers of other cells. Intracellular molecules, e.g., histamine, were gradually increased in a mouse bone marrow-derived mast cell by a maturation process. A single cell was captured by a microsuction pipette, and the mass spectra of intracellular histamine were measured directly. Finally, the time course of the intracellular molecular contents and the maturation stage from a single cell were estimated.

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A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, ∼12.5 μm) with a very small volume (∼2 μl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, response time, sensitivity and selectivity for measurements of L-glutamate. The currents at 0 V vs. Ag/AgCl increased linearly with the L-glutamate concentration from 10 to 150 μM for in vitro and in situ calibrations. The reponse was highly selective to L-glutamate over ascorbate, depamine, serotonin and other amino acids. The detection of L-glutamate in the extracellular fluids of different regions of mouse hippocampal slices under stimulation of KCl was demonstrated.

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An electrochemical in vivo flow-injection system with an on-line microdialysis sampling is proposed for the simultaneous monitoring of L-lactate and glucose in rat brain. In the first stage of the operation, the dialysate from the microdialysis probe is delivered to a sample loop of the six-way autoinjector by perfusing Ringer’s solution for 80 s at 5 μl min^-1. In the second stage, the dialysate collected in the sample loop is automatically injected for 10 s into the flow-injection line. Injected dialysate is split into two streams and two portions pass through two channels with two different immobilized enzyme reactors(glucose oxidase and lactate oxidase immobilized reactors) to produce hydrogen peroxide from glucose and L-lactate in the dialysate. After a subsequent confluence of the streams, produced hydrogen peroxide can be detected amperometrically at a downstream poly(1,2-diaminobenzene) film-coated platinum electrode, without any interference from oxidizable species and proteins present in the dialysate. Because each channel has a different residence time, two peaks are obtained. The first peak corresponds to L-lactate and the second peak to glucose. The peak current is linearly related to the concentrations of L-lactate between 0.2 and 10 mM and glucose between 0.1 and 20 mM. The present method can be successfully applied to the simultaneous in vivo monitoring of L-lactate and glucose in rat brain. The analytical speed is 45 dialysates h^-1.

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High-resolution single photon emission computed tomography (SPECT) provides a unique capability to image the biodistribution of radiolabeled molecules in small laboratory animals. Thus, we applied the high-resolution SPECT to in vivo imaging of the brain dopaminergic neurotransmission system in common marmosets using two radiolabeled ligands, [¹²³I]2β-carbomethoxy-3β-(4-iodophenyl)tropane (β-CIT) as a dopamine transporter (DAT) ligand and [¹²³I]iodobenzamide (IBZM) as a dopamine D₂ receptor (D₂R) ligand. Specific images of the striatum, a region with a high density of dopaminergic synapses, were obtained at 240 min and 60 min after injection of [¹²³I]β-CIT and [¹²³I]IBZM, respectively. Furthermore, a significantly low accumulation of [¹²³I]β-CIT in the striatum was observed in MPTP-treated animals compared with results for a control group, and a similar accumulation in the control group was observed with the pretreatment of deprenyl in the MPTP-treated animals. However, the striatal accumulation of [¹²³I]IBZM showed no changes among the control, MPTP-treated, and deprenyl-MPTP-treated groups. These SPECT imaging results agreed well with those of DA concentration and motor behavior. Since MPTP destroys nigrostriatal dopamine nerves and produces irreversible neurodegeneration associated with Parkinsonian syndrome, SPECT imaging data in this study demonstrated that deprenyl shows its neuroprotective effect on Parkinsonism by protecting against the destruction of presynaptic dopamine neurons.

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We describe here a method of affinity capillary electrophoresis in which oligodeoxynucleotide(ODN) was immobilized onto the inner surface of the capillary. The immobilized ODN functioned successfully as an affinity ligand for sequence-based DNA separation. Six-or 12-mer ODN with a sequence complementary to one of the c-K-ras gene was used as an immobilized ligand. When the 12-mer ODN was used, the detection peak for the complementary ODN disappeared selectively, while the single-base mutant was detected as useal. In contrast, when the 6-mer ODN was used as the affinity ligand with a mixture of the complementary ODN and its single-base mutant, it was possible to detect both as completely separate peaks. That is, the separation mode was dependent on the base number of the immobilized ODN used as an affinity ligand.

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A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40°C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.

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Sialyl Lewis X (SLEX) antigen, Neu5Acα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, plays important roles in cell-to-cell interaction: for example, the E- and P-selectin-mediated influx of SLEX expressing leukocytes into inflamed areas. A human hepatocellular carcinoma cell line, HepG2 cells, was highly expressed SLEX on secreted glycoproteins and cell surface, in contrast with HuH-7 cells. We identified SLEX expressing glycoproteins in HepG2 cultured medium by two-dimensional polyacrylamide gel electrophoresis, followed by in gel digestion and peptide mass fingerprint using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS), including transferrin, α₁-antitrypsin, α₂-HS glycoprotein and β₂-glycoprotein. We analyzed N-glycans of these glycoproteins by MALDI-TOFMS in combination with exoglycosidase digestion; our results indicate increases in poly-fucosylated and high-branched N-glycans. High α1,3-fucosylation in glycoproteins would be caused by increased expression of α1,3-fucosyltransferase activities in HepG2 cells.

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The following procedures were established in order to develop useful degradation enzymes of glycoconjugate for developing postgenome and postproteome research: (1) Enzyme activity with a short time reliability was measured using small amounts by HPLC. (2) The structures of the sugar chains liberated from the glycoconjugate were non-destructively analyzed using small amounts of sugar chains only by 1D ¹H-NMR and H-H COSY spectrometry and a computer simulation of the spectrum. (3) The conformations of the sugar chains liberated from a glycoconjugate in aqueous solution were estimated using 1D ¹H-NMR and H-H COSY spectrometry and the anisotropic effect. Endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 developed using the above methods transferred the sugar chain to sugars and peptides; therefore, it was also an effective enzyme when synthesizing sugar chains and glycopeptides.

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On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0×10^-8 M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.

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We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luniferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03×10^-20 and 2.05×10^-20 mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.

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Immunoassay is widely employed as a highly sensitive, specific analytical method for hormones and drugs in biological samples. A technique utilizing capillary electrophoresis with laser-induced fluorescence detection was examined based on the reaction process of these immunoassays in order to develop a protocol characterized by high sensitivity and high speed. The conditions of the antigen-antibody reaction and capillary electrophoresis were variously examined using fluorescein-labeled methotrexate and the antibody of methotrexate. As a result, the immunoassay could be completed within a few minutes. Moreover, detection in the pg range could be accomplished. The sensitivity corresponded to that of radioimmunoassay. A simultaneous multi-component analysis of the immunoassay is also possible due to the high resolving power of capillary electrophoresis. In this study, the possibility of a simultaneous analysis of methotrexate and vancomycin was also investigated.

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The speciation of diverse elements in salmon egg cell cytoplasm was performed by a surfactant-mediated HPLC/ICP-MS hyphenated system. In the present experiment, an ODS column coated with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), which is a zwitterionic bile acid derivative, was employed as a surfactant-mediated separation column, and ICP-MS was used as an element-selective detector. The present surfactant-mediated HPLC allowed us to separate large and small molecules within 10 min; large molecules, such as proteins, were eluted within 2.5 min, while small molecules were eluted after 2.5 min, but within 10 min. In the present experiment, Fe, Cu, and Zn in egg cell cytoplasm were observed mostly in species with large molecular weights, indicating that these elements are contained as metalloproteins or metalloenzymes in egg cell cytoplasm. On the contrary, it was found that P, S, Mo, and halogens in egg cell cytoplasm were contained as small molecules or inorganic ions. The major species of P in egg cell cytoplasm was identified as the phosphate ion (PO₄^3-). Molybdenum, Cl, and Br in egg cell cytoplasm were molybdate (MoO₄^2-), chloride (Cl^-), and bromide (Br^-) ions, respectively.

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The chemiluminescence (CL) intensities of various indole derivatives substituted with a glyoxylyl group at the 3-position and a hydroxyl group at the 5-position of the indole ring were compared upon the addition of H₂O₂ in alkaline media. The CL intensites of 3-indoleglyoxylyl chloride, 3-indoleglyoxylic acid, 5-hydroxyindole and 5-benzyloxyindole in CH₃CN were 5.9-, 48-, 5.9- and 3.3-fold stronger than that of 3-methylindole. A lasting CL of 3-indoleglyoxylyl chloride was found. Under appropriate conditions, the CL emission reached a maximum within 10 min after the addition of H₂O₂ in the presence of NaOH, and the intensity was retained for 25 min. One of the final products via the CL reaction of 3-indoleglyoxylyl chloride was indole-3-carboxylic acid. 3-Indoleglyoxylyl chloride emitted light by decompositions via both dioxetane and dioxetanedione. An enhancement effect of β-cyclodextrin and bovine serum albumin on the CL of 3-indoleglyoxylyl chloride was also found.

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We constructed an assembled-type MgF₂ cell that can function under a high vacuum (10^-4 Pa), and is capable of measuring the vacuum-ultraviolet circular dichroism (VUVCD) spectrum in a wavelength region down to 140 nm for aqueous solutions. Its path length can be adjusted by various spacers in the range from 1.3 to 50 μm. The temperature-control unit of the cell was also constructed with a Peltier thermoelectric element to keep the temperature of a sample within an accuracy of ±1°C in the temperature range from −30 to 70°C. The optical cell and the temperature-control unit were confirmed to have good performance by monitoring the VUVCD spectra of ammonium d-camphor-10-sulfonate and myoglobin aqueous solutions. This cell is available not only for VUVCD spectroscopy, but also for vacuum-ultraviolet absorption measurements.

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Complex formation studies of L-glutamate with aluminum(III) ion were conducted in acidic aqueous solutions (pH 2.0-5.5) by means of pH-metric titration and multinuclear (¹H, ¹³C and ²⁷Al) NMR techniques. The following results were obtained: (1) Al could weakly coordinate with Glu to form various mononuclear 1:1 (AlLH²⁺, AlL⁺, AlLH_-1) species and dinuclear 2:1 (Al₂L⁴⁺) species in acidic aqueous solutions, which somewhat agreed with previous findings. (2) The multi-NMR spectra of Al-Glu and Al-Asp strongly suggest that, besides negatively charged carboxylate donors (-COO^-, -COO^-), the amino group of Glu can participate in the binding of Al in the AlL⁺ and AlLH_-1 species in the case of its deprotonation, which rather agreed with the case of Al-Asp. (3) These tridentate five-+seven-membered joint chelate (-COO^-, -NH₂, -COO^-) complexes exhibit an enhanced stability, which can help to better understand the biological studies that Al-Glu could cross the erythrocyte membrane and the blood-brain barrier (BBB) and be deposited selectively in various brain regions, particularly in the cortex. It will also help to intrinsically understand the Al’s role in the biological transamination system, which is a very important process in all living things.

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The effects on humans inhaling the fragrance of essential oils were examined in terms of a sensory test, a multi-channel skin thermometer study and a portable forehead surface electroencephalographic (IBVA-EEG) measurement. The essential oils examined in this study were those of basil and peppermint, because our previous sensory test had indicated an opposite effect of these essential oils when mental work was undertaken; the inhalation of basil produced a more favorable impression after work than before work, whereas peppermint produced an unfavorable impression under these circumstances. For subjects administered basil or peppermint before and after mental work using an inhalator, a series of multi-channel skin thermometer studies and IBVA-EEG measurements were conducted. Using such paired odorants, our results showed that when compared between before and after mental work assigned to subjects: (1) the inhalation of basil, in which a favorable impression was predominant on the whole in terms of the sensory evaluation spectrum, was shown to be associated upward tendency in finger-tip skin temperature; (2) whereas these situations were opposite in the case of peppermint, in which the reversed (unfavorable) feature in sensory profiling was accompanied by a decrease in the magnitude of beta waves and a decrease in the finger-tip skin temperature both based on Welch’s method, even at p < 0.01, implying a decreasing propensity of the aroused state and of the arousal response. The elucidation of such sensory and physiological endpoints of paired odorants would be of primary importance for human chemoreception science, because these are only rarely recorded during the same experiments, and this paradigm is highly informative about non-verbal responses to odorants.

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The major-to-ultratrace elements in human bone-marrow fluid were determined by ICP-AES (inductively coupled plasma atomic emission spectrometry), and ICP-MS (inductively coupled plasma mass spectrometry). The bone-marrow fluid sample was centrifuged prior to acid digestion to exclude the bone piece from bone marrow, and then digested with nitric acid. As a result, 20 elements could e determined over the concentration range from 1610 μg g^-1 for Na to 0.00043 μg g^-1 for W. It was found that Fe, Zn and Sb were enriched by ca. 264-, 7- and 15-fold, respectively, in bone-marrow fluid, compared to those in human blood serum. Alkali metals (K, Rb, Cs), except for Na, were also significantly enriched in bone-marrow fluid. Furthermore, the concentrations of various elements, such as Fe, P, Al, Zn, Cu, Se, Zr, Sn, Ag and W, were significantly higher than those in open seawater.

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We improved an analytical method for determining trace amounts of bisphenol A (BPA) in urine. BPA was subjected to enzymolysis and then to solid phase extraction with a C₁₈ cartridge. The extract was eluted with methanol, and the eluate was concentrated under a nitrogen stream, and then pentafluorobenzylized in an alkali solution. The obtained pentafluorobenzylized compound was purifed using a florisil cartridge, followed by a determination using NCI-GC/MS. This method exhibited an excellent selectivity and reproducibility with a determination limit of 0.1 ng/ml.

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A needle-shaped sampler/reactor was developed for an electrochemical enzyme immunoassay with the direct sampling of living sample blood. This device was evaluated using IgG determination chemistry. Antibodies were immobilized on an inner wall of the sampler/reactor. Incubation for the enzyme reaction was not needed because this reactor was very small (250 μm in diameter). The analysis was conducted within 15 min in the simplest protocol including the reactor refreshment. The limit of detection was 3 pg, and 20 attomol in the most sensitive protocol. Furthermore, the sampling of a solution contained in an agar block and a whole-blood analysis were demonstrated.

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A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC₅₀) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.

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Changes in the chemical structures of morphine and codeine in the presence of hydrogen peroxide, a major component of hair dye and decolorant treatments, were examined with high-performance liquid chromatography/mass spectrometry (LC/MS). A mixture of morphine and hydrogen peroxide solution, after incubation at 39°C for 24 h, produced two reaction products (hydroxymorphines). When codeine was used in place of morphine, one reaction product (hydroxycodeine) was produced, in which the benzene ring was hydroxylated.

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A simple method to label oligosaccharides with a multifunctional fluorescent group was developed. Oligosaccharides were quantitatively labeled at their reducing termini with pyrene butanoic acid hydrazide. The pyrene-labeled oligosaccharides were successfully applied to fluorescence polarization measurements and ELISA at picomole quantity, which was not previously reached by other procedures. this labeling method should prove to be useful in a variety of aspects in glycobiology.

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A peptide fragment of a non-amyloid-β component (NAC(1-13)) was studied by CD and electron microscopy. Typical amyloid fibrils were observed by EM in a solution of NAC(1-13). In addition to the β-structural CD band in the far-UV region, a novel CD band near 285 nm was observed in a peptide solution of NAC(1-13). Taking the NAC(1-13) to contain neither an aromatic amino acid residue nor cystine into account, the CD band can be attributed to amyloid fibrils of NAC(1-13).

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For the requirement of a high, fast and sufficient technology to suit the needs of 21st century biotechnology, the separation range of a microchip electrophoresis system was studied. Two DNA fragments on the human Y-chromosome, SY594 (82 bp) and 12f2 (88 bp), were successfully separated with a reproducibility of 1.9% and an accuracy of 2.8%. Then, a mixture of 10 DNA markers ranging from 61 bp to 189 bp was successfully separated with high resolution. All of these results demonstrate the superiority of microchip electrophoresis as a tool for 21st century bioseparation.

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We developed DNA-conjugated polymer for DNA chip fabrication. A 30 mer probe DNA disulfide bridges were covalently attached to the polymer side chain. The DNA-conjugated polymer can be specifically adsorbed on a gold substrate surface by a self-assembly technique. The interaction between fully matched DNA and DNA-conjugated polymer was investigated by surface plasmon resonance (SPR) technique. The DNA-conjugated polymer-modified gold surface highly recognized fully matched DNA, rather than unmatched DNA. Therefore, DNA-conjugated polymer can be used for novel DNA chip fabrication.

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A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the λ phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.

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We have recently reported on dodecamer peptides (HPPMDFHKAMTR, CHPQPLKSRNPL) which recognize 52 - 58th and 197 - 203rd amino acid sequences of glucose oxidase (GOx) by screening via a phage random peptide library. In this study, a side-chain protected peptide monomer (PPM) was synthesized using two peptides (HPPMDFHKAMTR, SHPQPLKSRNPL) and acryloyl chloride. The peptide-conjugated polymer (PCP) was copolymerized with PPMs and N,N-diethylacrylamide (DEAA). The affinity of PCPs to GOx was estimated using surface plasmon resonance detection. This study suggests that PCP is a valuable molecular recognition biomolecule.

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