BUNSEKI KAGAKU Abstracts

In a crisis and the consequent management of biological and chemical warfare terrorism using anthrax or sarin, for instance, on-site monitoring and detection are necessary to protect against terrorism and the minimization of damage. The detection performance required for on-site detection can be determined in consideration of the fatal concentration and the minimum infectious dose, as well as their manifestation time. We evaluated the detection performance of commercially available on-site equipment for detecting biological and chemical warfare agents: detection paper, gas detection tube, flame photometric detector, photoionization detector, ion mobility spectrometer, surface acoustic wavelength detector, Fourier-transformed infrared spectrometer, mass spectrometer, ATP bioluminescence detection kit, flow cytometer, and immunostrip, using authentic agents and simulants. There was no detection equipment that fulfilled all properties of the agent species detected, detection sensitivity, detection accuracy, response time and operation easiness. By adopting new detection technologies, a monitoring tape method, enabling the sensitive and quick detection of gaseous chemical warfare agents, and counter-flow introduction atmospheric pressure ionization mass spectrometry, enabling sensitive and real-time monitoring of volatile and nonvolatile chemical warfare agents, and biosensor using sugar chain molecular recognition tool for biological toxins, the combination of existing high-performance on-site detection equipment should provide simultaneous and rapid detection of all biological and chemical warfare agents.

Although Maillard reaction (glycation) between protein and glucose has been thoroughly investigated, we have found that it is possible for glycation to occur between lipid and glucose. Our finding hypothesizes that under a hyperglycemic condition (i.e. diabetes), membrane phospholipid may be abnormally glycated in vivo, contributing to pathogenesis. In evaluating the hypothesis, we demonstrated that glycated phospholipid caused oxidative stress (lipid peroxidation) and angiogenesis, and developed a preparation method of pure glycated phospholipid as well as its highly sensitive assay. In this paper, we introduce our analysis technique of glycated phospholipid, give evidence of the occurrence of lipid glycation in a biomembrane, and discuss the relation of lipid glycation to lipid peroxidation and cell functional disorder.

Electrical DNA detection, using a gold nanoparticle modified by a thiolated probe DNA, based on the conductivity of double-strand DNA is described. In this method, probe-modified Au particles are immobilized between the electrodes (quantity of modified DNA on the Au surface, 16 pmol/cm²). Two different probes (12 mer) are hybridized by the addition of target DNA (24 mer) in which the formation of a Au particle-24 mer double-strand DNA-Au particle is structured between the electrode gap. The resistance decrease was observed immediately after the addition of target DNA, which became stable within 30 s with high S/N ratios (＞10). The resistance change of the Au particle film depended on the number of mismatching base pairs (bp) in the DNA sequence. The difference in the resistance change was obtained between complementary (0.31 ohm) and 2 bp mismatch (0.18) DNA, which indicates that the Au particle film rapidly discriminates only a 2 bp mismatch in the sequence.

A convenient electrochemical method for the assay of α-glucosidase was developed using hydroquinone-α-D-glucopyranoside (α-arbutin) as a substrate. The concentration of p-hydroquinone produced by the hydrolysis of α-arbutin was measured by amperometry with a membrane-covered glassy carbon electrode at 0.4 V. By the addition of α-glucosidase to a buffer solution containing α-arbutin, the current began to increase linearly. The initial rate of hydrolysis was calculated from the slope of the current-time curve. The initial rate was increased with increasing the concentration of α-arbutin, and tended to approach the saturation value, whereas it was proportional to the concentration of α-glucosidase. The Michaelis constant (K_m) for α-arbutin by α-glucosidase was 0.45 mM. There was the good correlation between the enzymatic activity of α-glucosidase determined by the present method and that determined by a spectrophotometric method using p-nitrophenyl-D-glucopyranoside as a substrate. Compared with the spectrophotometric method, the electrochemical method has advantages of being free from the influence of turbidity and coloration of a sample solution. Therefore, the activity of α-glucosidase in a solution extracted from rice koji could be measured without any filtration treatment.

A simple and rapid FIA method for measurement of quenching effects of grape seed extracts and polyphenolics against reactive oxygen species (ROS) was developed. This method is based on the determination of luminol chemiluminescence generated with ROS such as hydroxyl radical (^･OH), singlet oxygen (¹O₂), peroxynitrite (ONOO^－) and superoxide anion (O₂^－). More than 1 sample/min could be measured with satisfactory precision. The total reagent amount in this method could be reduced to one tenth of that for our previous batch method. The grape seed extracts A and B were successfully evaluated by the proposed method. The EC₅₀ of cyanidin as a representative polyphenolic against ^･OH, ¹O₂, ONOO^･ and O₂^－ were 50±4, 148±8, 36±4, and 95±8 ng/assay (n＝3), respectively. Except for chalcone, the other polyphenolics such as pelargonidin, delphinidin and trans-resveratrol also showed the high quenching effects against ROS.

Many organic compounds emit chemiluminescence by auto-oxidation. Meanwhile, we reported that oxygen molecules were adsorbed to various compounds and could emit ultraweak chemiluminescence (CL) by heating in a nitrogen atmosphere. However the details of the bonding site and the state of oxygen are still unknown. In this study, we measure the CL intensity and the spectrum from compounds that contained ether oxygen, and investigated the difference of the amount of adsorbed oxygen and the bonding state in different compounds. Luminescence from ethylene glycol (EG) and diethylene glycol (DEG) was nearly zero. However, triethylene glycol (TEG) and polyethylene glycol (PEG) exhibited strong luminescence. All ethylene glycols have two hydroxyl groups at the ends, and TEG has two ether oxygen atoms, DEG has one, and EG has none. Thus oxygen molecules could be bound to two ether oxygen atoms of TEG. Luminescence from ribose, which has an ether oxygen atom in the molecule, showed a characteristic peak and the decayed above the melting point. Based on the observed data, oxygen molecules might be binding to ether oxygen atoms between two molecules in the solid condition.

Fluoroquinolones (FQs) are an important group of synthetic antibacterial, which are widely used to treat human and veterinary diseases. In recent years, FQs residues were detected in several samples. An enzyme-linked immunosorbent assay (ELISA) is useful to determine many samples simultaneously. However, there is a problem of not having qualification because of cross-reactivity. In the present study, we developed an analytical method using HPLC/FL for a comparative study with ELISA. Moreover, an HPLC/FL system was applied to the determination of FQs residues in meat samples. The LOD and LOQ of HPLC/FL were 2 ng/g and 10 ng/g for enrofloxacin, respectively. The extraction recovery of enrofloxacin spiked concentration of 50 ng/g was 107.8％. The HPLC method and an enzyme-liked immunosorbent assay for the analysis of fluoroquinolones in meat samples were compared. FQs were detectable in meat samples added FQs standard by HPLC/FL and ELISA. Moreover, there was a correlation between HPLC/FL and ELISA. In conclusion, ELISA may be useful to rapid monitoring of residues for FQs instead of HPLC/FL, which requires analytical times of 120 min in meat samples.

The sample preparation technique of stir bar sorptive extraction (SBSE) was used for the determination of polycyclic aromatic hydrocarbons (PAHs) in river water samples. The 100 ml river water sample and 10 ml of methanol were placed in a 125 ml vial. A stir bar coated with 126 μl of polydimethylsiloxane was added to the sample, and the extraction was performed at 800 rpm for 3 hr. After the extraction, the stir bar was placed into a 250 μl insert of a 2 ml vial filled with 200 μl of acetonitrile. Liquid desorption was performed with sonication for 15 min. The extract solution was analyzed by high-performance liquid chromatography with a fluorescence detector (HPLC-FL). Once SBSE was optimized, validation methods such as linearity (r＞0.999), detection limits (0.6〜6.0 pg/ml) and quantitation limits (2.0〜20 pg/ml) were performed. The average recoveries of PAHs were higher than 94.8％ (RSD: 1.9〜8.6％). This simple, accurate, sensitive and selective analytical method may be used in the determination of trace amounts of PAHs in river water samples.

The purpose of this study is to find the exposure sources of perfluorochemicals (PFCs). We have developed a method for measuring 4 PFCs (perfluorooctanesulfonate; PFOS, perfluorooctanoic acid; PFOA, perfluorononanoic acid; PFNA and perfluorohexanesulfonate; PFHxS) in house dust using supercritical fluid extraction (SFE) and based on high-performance liquid chromatography/tandem mass spectrometry. The mean extracted recovery assessed at two different concentrations (50 and 250 ng/g in house dust) was more than 97.9％. The assay was linear over the range 2.5〜500 ng/g. The method detection limits were assessed as being 0.58 ng/g to 0.72 ng/g. We determined the concentrations of PFCs in 20 house-dust samples for investigating of the exposure source. The compounds were detected in all dust samples and the ranges were 7.0〜41 ng/g for PFOS, 18〜89 ng/g for PFOA and 5.5〜69 ng/g for PFNA. PFHxS was determined in 8 samples (2.5〜5.5 ng/g). Based on our experiments, house-dust might be one of the human exposure sources for PFCs. The developed method can be applied to the determination of PFCs in house dust samples for monitoring human exposure sources.

For the systematic analysis of the proteomes of microorganisms whose genomic nucleotide sequences have been analyzed, a method for protein separation and purification using anion-exchange column chromatography was developed. Generally, proteome analysis has a problem that membrane proteins become insoluble during the sample preparation steps, and are difficult to detecte. We adopted the use of an anion-exchange column chromatography in the first step of protein separation, and optimized the method to allow the direct injection of whole cell lysates without any preprocessing. After removing soluble proteins, insoluble proteins retained on the column were subjected to in-column enzymatic digestion, followed by elution. The method was applied to the proteome analysis of Aeropyrum pernix K1, an aerobic hyper-thermophilic archaeon, and proteins were identified by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Thus, a total of 10 novel membrane proteins were detected and identified. Consequently, this method seems to be effective to detect water-insoluble membrane proteins that had never been detected by other conventional methods.

An intracellular signal transduction system involved in many kinds of proteins regulates various cellular events, such as cellular proliferation, differentiation and cell death, responding to the outer environment. Among those proteins, protein kinases are one of the most important class of proteins. It has been reported that abnormal activities of protein kinases are related to various diseases. Therefore, an analysis of protein kinase activity would be useful for diagnosis and drug screening. Herein, we describe the detection of on-chip phosphorylation using a peptide chip by fluorescence imaging. In this work, we selected a cAMP-dependent protein kinase (PKA) as a model, and quantitatively evaluated the substrate specificity for PKA using a peptide chip. Moreover, we attempted to detect intracellular kinase activity. Since src correlates with breast cancer, we measured the src activity in MCF-7 human breast cancer cell lysate. Consequently, with our peptide chip we could detect the src activity in MCF-7 cell lysate. Therefore, this method could be applied to diagnosis, drug discovery and screening.

Electrochemical oxidation on a nucleic acid base, such as guanine, adenine, thymine, cytosine, was examined; only guanine could be oxidized on a gold electrode. Because deoxyguanosine, deoxyguanosine monophosphate were also oxidized on the electrode, guanine in DNA would be oxidized on the electrode. To prove DNA oxidation, thiolated-single strand DNA (ssDNA) was immobilized on a gold electrode, and electrochemical measurements were performed: differential pulse voltammetry (DPV) and chronoamperometry were examined. The voltammogram of DPV on an ssDNA-modified electrode had an oxidation peak at ＋1.17 V vs. Ag/AgCl. The oxidation current was caused by the oxidation of guanine in the DNA-base. The electrical charge difference between a modified electrode and a bare electrode on chronoamperometry agreed with the guanine amount of ssDNA on gold, which was obtained from the result of a quartz crystal microbalance.

Scanning electrochemical microscopy (SECM) has been applied to enzyme immunoassay for the detection of C-reactive protein (CRP). The immunocomplexes of the sandwich type with horseradish peroxidase (HRP) labeling were constructed at the microspots of an anti-CRP IgG antibody fabricated on a hydrophobic glass substrate by capillary microspoting. In the presence of ferrocenemethanol (FcOH) as an electron mediator and hydrogen peroxide as a substrate of HRP, the oxidized form of FcOH (Fc^＋OH) was generated at localized areas corresponding to the microspot of immunocomplexes by an enzymatic reaction of captured antibodies with HRP label. The reduction current of Fc^＋OH was detected with a microelectrode at 0.05 V vs. Ag/AgCl and mapped by scanning the microelectrode to view SECM images of the spots for CRP. An amperometric determination of CRP was also performed using the microelectrode positioned at 10 μm above the microspots. Relationships between the reduction current in SECM images and the concentration of CRP, were obtained in the range of 0.1 ng/ml to 100 ng/ml. A system for multi-samples measurements has been developed using amperometric determination and antibody array chips.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is beginning to play an important role in a rapid discrimination of bacteria, where ribosomal proteins composed of more than 50 subunits is used as biomarkers. In this study, this technique was applied to the maintenance of bacterial culture collections. It was confirmed that most of the biomarker peaks could be observed in the whole cell lysate of model lactic acid bacteria, which was prepared simply by bead-beating and centrifugation. The mass differences of specific ribosomal subunit proteins observed on the MALDI mass spectra allowed us to discriminate two lactic acid bacteria at a subspecies level difference (Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris). By comparing the MALDI mass spectrum of the stored culture of Lc. lactis subsp. lactis with that of the pure culture as a reference, it could be easily found that the stored culture was cross-contaminated with other lactic acid bacteria.

The purpose of this research is the establishment of ELISA for measuring dioxins in environmental samples, such as house dust, soil, exhaust gas and fly ash. Since we have developed dry-type plates in order to put the ELISA system into practical use, the basic performance, such as the cross-reactivity, linearity and the correlation between the conventional GC/MS values and the ELISA values, were studied. The dry plates could be stored for about one year in a refrigerator. Furthermore, a good correlation was observed between the dioxin quantities by ELISA and those by GC/MS in exhaust gas, fly ash, soil and house dust samples. The developed ELISA system indicated usefulness as a toxicity evaluation method for dioxins in environmental samples.

A fading phenomenon of laver arose in the Ariake Sea, Japan from the end of 2000 to the beginning of 2001. It was said that consumption of the major nutrient (N, P. etc.) of sea water by the large phytoplankton caused this phenomenon. In the present study, the relationship between the fading phenomenon of laver and metal elements in sea water was examined. It was found that the amount of essential trace metal elements (especially Fe, Mn and Zn) of the faded laver was less than those of normal laver. In addition, the concentration of these metals in sea water collected near the area around the color-faded laver decreased extremely compared with previous data. From these results, it was deduced that not only the major elements (N and P), but also trace essential elements (Fe, Mn and Zn), could be consumed by phytoplankton. Therefore, the deficiency of these elements could be the major reasons for the fading phenomenon of laver in the Ariake Sea.

A phenylboronic acid-appended azo dye (Azo-PBA) binds saccharides and shows a color change at neutral pH. We investigated the effects of poly(allylamine) (PAA) on the binding property of Azo-PBA. The UV-visible absorption spectra of Azo-PBA were changed in the presence of PAA, depending on the concentrations. This result indicates the formation of Azo-PBA aggregates. In the absence of PAA, the binding constants of Azo-PBA to D-glucose and D-fructose were calculated to be 1.4 and 75 M⁻¹, respectively. In the presence of PAA, the binding constant to D-glucose was 130 M⁻¹, which was 93-times higher than the original binding constant, while the binding constant to D-fructose was increased only 3.3-times. These results show that the coexistence of PAA can selectively enhance the binding ability of Azo-PBA to D-glucose. However, it was found that a small amount of D-fructose interfered in the measurement of D-glucose.