BUNSEKI KAGAKU Abstracts

A nanometer-sized gold nanoparticle (AuNP) – glucose oxidase (GOx) hybrid has been prepared by a single-step procedure. This paper examines the roles of AuNPs in the enzyme activity of the hybrids by considering electrochemical and fluorescence measurements. The immobilization of the hybrids on a glassy carbon electrode led to an increase in a current response of 2.5 times, compared to that of GOx. On the other hand, there was no difference in the fluorescence intensity based on the production of hydrogen peroxide through the enzyme reaction in between GOx and AuNP – GOx. These results indicate that AuNPs in the hybrid does not affect the enzyme activity, but promotes an electron transfer originated through an enzyme reaction to the electrode.

Novel water-holding separation materials (Betaine-resin and DAM-resin) having zwitter-ionic functional groups were synthesized by introducing sulfobetaine and diallylamine-maleic acid copolymer to a hydrophilic methacrylate base resin. These separation materials showed high water-holding ability equal to ion-exchange resins. Their extraction efficiencies of water-soluble compounds in acetonitrile solutions were evaluated by the solid-phase extraction method using cartridges packed with the materials. The materials showed high affinity for nucleobases, nucleosides and organic acids, and could be extracted quantitatively. The DAM-resin could quantitatively extract highly water-soluble solutes, such as glucose and glucosides. Therefore, the chromatographic properties for the water-soluble solutes on the DAM-resin were evaluated by HPLC. Because the logarithm of the retention factors (Log k) of the water-soluble solutes showed good correlation to their Log P_o/w (logarithm of octanol–water partition coefficient), it was recognized that the water-soluble compounds should be able to be partitioned into the water-rich layer associated with the zwitterionic moieties. On the other hand, the ionic solutes were retained by a mixed interaction mode combined with an electrostatic interaction. However, their retentions could be regulated by controlling the composition of the copolymer, the characteristic of salt in the mobile phase, and the mobile phase pH. Furthermore, a fibrous adsorbent (DAM-fiber) was successfully developed by the wet-spinning of viscose rayon with the DAM, and was characterized for the extraction and adsorption of water-soluble compounds. Further, the DAM-fiber also showed high decline rates for volatile acidic and basic substances in atmosphere, and demonstrated a high ability as an adsorption/removal material for odor substances.

The hydroxyl and carboxyl end-groups in poly(lactic acid) (PLA) were derivatized with trimethylsilylation or trifluoroacetylation to be detected by ¹H DOSY or ¹⁹F DOSY with enhanced sensitivity. The diffusion coefficients (D) for the derivatized end-groups of PLA in a chloroform-d solution were determined by ¹H and ¹⁹F DOSY analysis; the values agreed well with each other. The D values for the end groups were found to be larger than those for the methyl and methine groups in repeating units of the PLA. The relative intensity of the ¹H DOSY resonances due to the trimethylsilyl end-group and the main-chain methyl group allowed us to evaluate the dependence of the degree of polymerization on D. ¹H DOSY analysis of PLA treated with diphenylmethane diisocyanate (MDI) was also carried out to determine D for the junction unit in the PLA. The result clearly indicated that the molecular weight of the PLA increased by the reaction of MDI and the hydroxyl end-groups.

To decrease the amount of sample solution in ICP-AES/MS, we developed a new droplet direct injection nebulizer (D-DIN) system in which aqueous solutions were not nebulized, but injected into the plasma as a series of small droplets. The typical droplet volume is 700 pL, which that is about 100-times larger than the droplets in the mist from usual conventional nebulizers. In this study, for stable plasma generation under introducing large size droplets, we developed a free-running ICP generation system. To evaluate the frequency shift by changing the plasma impedance, the plasma generation conditions, such as the RF-power, plasma gas flow rate and carrier gas flow rate were changed and the oscillation frequency was measured. Upon increasing the RF-power, the oscillation frequency was increased to around 150 kHz for both Ar and He ICP. However, upon increasing the plasma gas flow rate, the frequency increased by 10 kHz in He ICP, and decreased by 20 kHz in Ar ICP. In addition, the frequency decreased by around 10 kHz Ar, and He ICP by increasing the carrier gas flow rate. These results suggested that this ICP generation system had sufficient following perfomance for fluctuation caused by large size sample droplet introduction into ICP.

For stable plasma generation under introducing large-size sample droplets to ICP, we designed and developed free-running ICP. In the system, the impedance shift of the plasma generated by introducing the droplet is immediately cancelled by a shift of the RF resonant frequency of the entire system. To evaluate the shift of the frequency caused by changing the plasma impedance with introducing 5 nL droplets, the frequency shift in the free-running ICP was measured. In the case of Ar-ICP, the frequency increased by 17.3 kHz from the base frequency, and then returned to the base frequency in 130 ms. The excitation temperature was about 3000 K decreased and then became about 4000 K increased rapidly. When the same measurement was performed in the case of He-ICP, the resonant frequency decreased by 3.4 kHz, and then returned in 24 ms, the excitation temperature was about 500 K increased and then 1000 K decreased. In addition, the frequency fluctuation was increased due to droplet volume in both cases. The cause of the frequency-shift characteristics was also discussed by using an electric circuit simulation. The results showed that the difference of the frequency sifts tendencies between Ar and He ICP can be explained based on the cappling system for RF-power energy transfer.

The influence of using plasticized poly(vinyl chloride) (PVC) membranes in the culturing of cells was examined in order to apply an array-type semiconductor calcium ion image sensor to the bioimaging of living cells. Two plasticized PVC membranes, CaHDOPP-DOPP and K23E1-NPOE, were prepared from optimal membrane contents. The plasticized PVC membranes were characterized by the potential response to calcium ion. The K23E1-NPOE membrane had a high sensitivity to a low concentration of calcium ion. The linear dynamic ranges of the K23E1-NPOE membrane and the CaHDOPP-DOPP membrane were 10⁻² – 5 × 10⁻⁷ M and 10⁻² – 1 × 10⁻⁵ M, respectively. The plasticizers leaked from the membrane to an aqueous solution were measured by gas chromatography. The leakage of DOPP was significantly detected. However, the leakage of NPOE was not detected, and the concentration was less than the detection limit. A aqueous solutions that kept long contact with the membranes were examined by the growth observation of HeLa cells. The first contact solutions were prepared by contact with the membranes for 6 hours at room temperature. The second contact solutions were prepared with the same membrane and under the same condition after the first contact solution was removed. The contact solutions never killed the cells. The contact solutions inhibited cell proliferation for 24 hours during the cultivation. In all of the contact solutions, however, all cells grew up to form a cluster after more than 48 hours, similar as in the controlled solution. The inhibition effect of contact solutions of CaHDOPP-DOPP was stronger than that of K23E1-NPOE. In addition, the inhibition effect of both second contact solutions was weaker than that of both first contact solutions. Probably, a conventional conditioning of plasticized PVC membranes is effective for reducing the inhibition effect of the cell proliferation. In conclusion, the K23E1-NPOE membrane was superior to the CaHDOPP-DOPP membrane to apply plasticized PVC membrane to Ca²⁺ imaging of living cells with array type semiconductor sensors.

A liquid chromatography tandem-mass spectrometry (LC/MS/MS) method has been developed for the highly sensitive determination of herbicides, pyraclonil, daimuron, pyrazosulfuron-ethyl and benzofenap, in river water. River water was filtrated with a glass fiber filter, followed by solid-phase extraction with a styrene-divinylbenzene copolymer with a nitrogen-containg polar group-methacrylate cartridge. Positive electrospray ionization (ESI-positive) was used to ionize herbicides, and the determination was performed by the selected reaction monitoring (SRM) method. The method detection limit (MDL) and the method quantification limit (MQL) are 0.78 ng L⁻¹ and 2.0 ng L⁻¹ for pyraclonil, 0.26 ng L⁻¹ and 0.67 ng L⁻¹ for dimuron, 0.44 ng L⁻¹ and 1.1 ng L⁻¹ for pyrazosulfuron-ethyl, 0.45 ng L⁻¹ and 1.1 ng L⁻¹ for benzofenap. The average recoveries from river water spiked with the herbicides; those concentrations were the same as MQL, and are 99%, 100%, 110% and 54%, respectively. All compounds except for pyrazosulfuron-ethyl were detected in river water collected after herbicide application.

For an intensity estimation of a free-introduction-decay (FID) signal in NMR spectra, the Prony method and high-order Prony method based on the generation model has been proposed. By using parameters that are obtained from the generation model, the intensity can be estimated with better accuracy than other methods, such as the “Integral Method” and the “Curve-fitting Method”. However, the accuracy of this method is still insufficient for the quantitative analysis of NMR spectra. We found that the accuracy of the high-order Prony method depends on the order of the generation model, and that the choice of an appropriate order realizes an accurate intensity estimation. In order to identify the appropriate order, we introduced a “selective reconstitution evaluation” (S.R.E.) into the high-order Prony method. A simulation using the extended method achieved an intensity estimation with an error below 1% in the case of a signal-to-noise ration of 100. The method was applied to an actual estimation of the intramolecular integral ratio of Acetaminophen. It was confirmed that the intensity can be estimated within an error of 1%.

A microwave digestion method for the determination of bromine in polyethylene (BCR 680 and 681) and peach leaves (SRM1547) by inductively coupled plasma mass spectrometry (ICP/MS) has been investigated. In the microwave digestion system, a sample of 0.1 – 0.3 g was weighed and 7 mL of nitric acid and 1 mL of hydrogen peroxide were added in a quartz insert. The insert was used within the standard TFM vessel which 6 mL of 10% hydrogen peroxide was added. The accuracy of the digestion method was evaluated. The recovery and the standard deviation (n = 3) for the bromine tested was 109% and 9.4%. The results obtained by the present method were in good agreement with the certified values.