Laser scanning confocal microscopy (LSCM) coupled to a constant volume flow-pressure measuring system is introduced as a new technique for the quantitative measurement of fluid flow across porous materials. Such processes are ubiquitous from the life sciences to materials science and the methodology herein could find widespread application. The methodology has been applied to the detection of fluid flow through human dentine, in-vitro, and in the assessment of occlusion actives. Dentine is a calcareous material sandwiched between the pulp and enamel in the tooth structure that contains tubules which traverse dentine in the pulp to enamel direction. The tubules become patent during enamel erosion or gum recession, leading to dentinal hypersensitivity. Understanding the nature of fluid flow is important, as a pressure gradient exists across dentine in-vivo and this has implications for the development of suitable treatments. The methodology described herein firstly allows a ready assessment of the general efficacy of treatments via hydraulic permeability measurements. Second, LSCM images allow the nature of the flow process and the mode of action of the treatments to be revealed at high spatial resolution. For the particular case of dentine, we demonstrate how the method allows candidate treatments to be compared and assessed.

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Theoretical equations for a particle size-determination method by applying an attenuated-total-reflection (ATR) technique were obtained. Based on these equations, the ATR-spectral intensities were calculated as functions of the sample amount and on the particle size for various choices of the experimental parameters. A mono layer region was more adequate for size measurements than a multiple-layers region. A prism and the incidence angle should be adequately selected according to the aims of the measurements. Experimental results of the sample amount dependences of the spectral intensities were almost consistent with calculations. The experimental results of size dependences showed the same tendency as those by calculations for SiO₂ and SiC; especially for SiC, they were in just accord with those by calculations in the case of a KBr prism. Each component of a mixed plural-kind-particles sample almost agreed in its spectral intensity with that of a corresponding single-kind-particles sample. The particle size-determination method, based on the ATR technique, was theoretically supported, and its characteristics were reveled.

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Data from X-ray powder diffraction (XRD) were subjected to a partial least-squares regression analysis (PLS) to build a calibration model for predicting the polymorphic content of carbamazepine (CBZ). The effectiveness of the PLS method in the construction of calibration models was analyzed by a scientific approach based on a regulation vector. CBZ forms I and III were characterized by differential scanning calorimetry (DSC) and XRD. Powder mixtures of forms I and III at various ratios (0 - 100% w/w; form III) were subjected to XRD. Five diffraction peaks were used for the peak-area method to compare with PLS. The results obtained by PLS had a better predictive accuracy compared to those of the peak-area method. The XRD-PLS method was established as a non-destructive, non-contact way to avoid the particle orientation effect based on statistical theory.

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A new type of dynamic light scattering method for the size measurement of nanoparticles was developed using a transmission grating. A sample cell was located behind the grating, and light was incident from the grating side. The scattered light by a solution with nanopariticles was mixed with diffracted light by the grating, and the mixed signal was detected; namely, the diffracted light was used as reference light for heterodyne detection. It was confirmed that the S/N ratio of the autocorrelation curve was 26-times improved by heterodyne detection. Furthermore, the S/N ratio was improved by setting the sample cell at the sample grating distance where the electromagnetic field is maximum due to the Talbot effect. Size measurements for several kinds of nanoparticles were demonstrated by this new method.

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Tetraazacyclododecane and tetraazacyclotetradecane derivatives bearing a spirobenzopyran and three carboxymethyl moieties, 1 and 2, and a diethylenetriamine derivative bearing a spirobenzopyran and four carboxymethyl moieties 3 were synthesized. The isomerization behaviors based on the spirobenzopyran moiety of these ligands were studied by UV-visible spectrophotometry in aqueous solutions containing various metal ions at neutral pH. These ligands formed stable 1:1 complexes with lanthanide ions, while the spirobenzopyran moiety was isomerized to its corresponding merocyanine form even under dark conditions. In aqueous solutions containing a lanthanide ion, the absorption spectra of 1 or 2 showed remarkable blue shifts, while absorbances at the maximum absorption wavelengths in the visible region were enhanced; such changes are attributable to the isomerization to the merocyanine form of the spirobenzopyran moiety. These results suggest that the phenolate anion of the merocyanine moiety interacts very strongly with a lanthanide ion bound by the complexing moiety because of the high charge density of lanthanide ions. In contrast, the absorbance of merocyanine form was decreased by the complexation of the macrocyclic ligand with transition metal ions, such as Cu²⁺ and Zn²⁺. This result indicates that macrocyclic ligands, 1 and 2, formed complexes with transition metal ions only by the aminocarboxylate moieties, and the phenolate ion of merocyanine moiety was not able to participate in the complexation. This conclusion was also demonstrated by density functional theory calculations.

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Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r² = 0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.

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Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H₂O₂ without lysis of liposomes. At a low concentration of H₂O₂, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H₂O₂. At a high concentration of H₂O₂, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H₂O₂ by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution.

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A novel design is described for an amperometric biosensor based on NAD(P)-dependent glucose dehydrogenase (GDH) combined with a plasma-polymerized thin film (PPF). The GDH is sandwiched between several nanometer thick acetonitrile PPFs on a sputtered gold electrode (PPF/GDH/PPF/Au). The lower PPF layer plays the role as an interface between enzyme and electrode because it is extremely thin, adheres well to the substrate (electrode), has a flat surface and a highly-crosslinked network structure, and is hydrophilic in nature. The upper PPF layer (overcoating) was directly deposited on immobilized GDH. The optimized amperometric biosensor characteristics covered 2.5 - 26 mM glucose concentration at +0.6 V of applied potential; the least-squares slope was 320 nA mM^-1 cm^-2 and the correlation coefficient was 0.990. Unlike conventional wet-chemical processes that are incompatible with mass production techniques, this dry-chemistry procedure has great potential for enabling high-throughput production of bioelectronic devices.

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A facile strategy of an amperometric biosensor for hydrogen peroxide based on the direct electrocatalysis of hemoglobin (Hb) immobilized on gold nanoparticles (GNPs)/1,6-diaminohexane (DAH) modified glassy carbon electrode (GCE) has been described. A uniform monolayer film of DAH was initially covalently bound on a GCE surface by virtue of the electrooxidation of one amino group of DAH, and another amino group was modified with GNPs and Hb, successively. The fabrication process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The proposed biosensor exhibited an effective and fast catalytic response to the reduction of H₂O₂ with good reproducibility and stability. A linear relationship existed between the catalytic current and the H₂O₂ concentration in the range of 1.5 × 10^-6 to 2.1 × 10^-3 M with a correlation coefficient of 0.998 (n = 24). The detection limit (S/N = 3) was 8.8 × 10^-7 M.

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The electrochemical properties of chloramphenicol at a boron-doped diamond thin-film (BDD) electrode were studied using cyclic voltammetry. The highest current response of chloramphenicol was obtained with phosphate buffer, pH 6 (0.1 M) in 1% ethanol. The relationship between the concentration of chloramphenicol and the current response was linear over the range of 0.1 - 10 mM (R² = 0.9990). The amount of chloramphenicol was analyzed by flow-injection analysis. A thin-layer flow cell equipped with a BDD electrode was used as an amperometric detector, and experiments were carried out at -0.7 V (vs. Ag/AgCl). The linear relationship between the current response and the concentration of chloramphenicol in the range of 0.1 - 50 µM (R² = 0.9948) and the limit of detection of 0.03 µM (S/N = 3) were obtained. This method has been successfully applied to the determination of chloramphenicol in sterile eye drops and milk sample by the standard addition method. The average recoveries of chloramphenicol in eye drops were 98.0%, and the average recoveries of chloramphenicol from spiked milk were 93.9 - 103%.

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A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 µm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35°C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2 - 150 µg/mL (R² = 0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 µg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.

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1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) is a very useful agent to form amide bonds (peptide bonds) in an aqueous medium. A simple and fast detection system was developed using the reaction with pyridine and ethylenediamine in acidic aqueous solution and spectrophotometric flow injection analysis. The absorbances were measured at 400 nm and the reaction was accelerated at 40°C. The calibration graph showed good linearity from 0 to 10% of EDC·HCl solutions: the regression equation was y = 3.15 × 10⁴x (y, peak area; x, % concentration of EDC·HCl). The RSD was under 1.0%. Sample throughput was 15 h^-1. This method was applied to monitoring the EDC·HCl concentration that remained after the anhydration of phthalic acid in water, esterification of acetic acid in methanol or dehydration condensation of malonic acid and ethylenediamine in water.

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An inductively coupled plasma mass spectrometer (ICP-MS) was used as an ion chromatographic detector for the speciation of iodine and bromine. Gradient elution using NH₄NO₃ at pH 10 allowed the chromatographic separation of ionic iodine (I^- and IO₃^-) and bromine (Br^- and BrO₃^-) species in less than 8 min. Effluents from the ion-exchange column were delivered to the nebulization system of ICP-MS for the determination of I and Br. The potentially interfering ³⁸Ar⁴⁰ArH⁺ and ⁴⁰Ar⁴⁰ArH⁺ at the bromine masses m/z 79 and 81 were significantly reduced in intensity (by approximately two orders of magnitude) by using 0.6 mL min^-1 O₂ as a reactive cell gas in the dynamic reaction cell (DRC). Moreover, the signal-to-background ratio at iodine mass m/z 127 increased significantly when O₂ was used as the reaction gas. The detection limits were in the range of 0.001 - 0.002 and 0.03 - 0.04 ng mL^-1 for various I and Br compounds, respectively, based on the peak height. The relative standard deviation of the peak areas for five injections of a 2 ng mL^-1 I^-, IO₃^- and 20 ng mL^-1 Br^-, BrO₃^- mixture was in the range of 3 - 4%. The concentrations of I and Br compounds have been determined in selected water and urine samples. The spike recoveries were in the range of 94 - 102% for all of the determinations. This method has also been applied to determine various I and Br compounds in an NIST RM 8435 whole-milk powder reference material and a seaweed sample obtained locally. A microwave-assisted extraction method was used to extract these compounds, which were quantitatively leached with a 10% mass/volume (m/v) tetramethylammonium hydroxide (TMAH) solution in a focused microwave field within a period of 6 min. The major components of I and Br in milk powder and seaweed were I^- and Br^-.

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A kind of new multi-layer adsorbent including Tenax TA/multi-walled carbon nanotubes (MWCNTs)/Carboxen 564 was developed for collecting volatile organic compounds. The adsorption and desorption efficiencies of 11 kinds of adsorbents (including Activated charcoal, Tenax TA, Carboxen 564, Chromosorb 101, Chromosorb 102, Chromosorb 103, Chromosorb 105, Porapak Q, GDX 301, XAD-2, and MWCNTs) were compared. By combining the advantages of Tenax TA, MWCNTs, and Carboxen 564, new type of multi-layer adsorbents was developed. The adsorption and desorption efficiency, the sampling reproducibility, and the effect of water were improved using multi-layer adsorbents, Tenax TA/MWCNTs/Carboxen 564. New multi-layer adsorbents were successfully applied to the determination of volatile organic compounds (VOCs) in ambient air.

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We have developed an analytical system to measure atmospheric ³He/⁴He ratios precisely by using a static vacuum operation mass spectrometer and an ultrahigh-vacuum purification line. After purification of the sample, helium was introduced into the mass spectrometer. Ion beams of ³He and ⁴He were measured at the same time by a double collector system. A resolving power of about 700 at the 5% level of the peak height was attained for the complete separation of ³He ions from those of HD and H₃. Repeated analysis of a sample calibrated against a standard showed a precision of about 0.2% error margin (2σ). This system has been applied to evaluating the ³He/⁴He ratio of He Standard of Japan (HESJ), and for the assessment of the atmospheric variation of latitude. The observed ³He/⁴He ratio of HESJ, 20.405 ± 0.040R_air (2σ) agrees well with the value of 20.408 ± 0.044R_air by Lupton and Evans, but is smaller than the recommended value of 20.63 ± 0.10R_air in a literature. Air samples collected from 45 to 20°N in the Far East showed a slight decrease of the ³He/⁴He ratio with latitude. Even though the variation is attributable to an experimental artifact, it may not preclude the possibility that the anthropogenic release of crustal ⁴He is significant in the high-latitude region of the northern hemisphere.

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Laser-induced photoacoustic spectroscopy was used in a quantitative analysis of Pu in HNO₃ medium. Plutonium was quantitatively oxidized to Pu(VI) using Ce(IV). The photoacoustic measurement of Pu(VI) with maximum absorption at 830.5 nm was subsequently performed to determine the concentration. The photoacoustic signal was linearly proportional to the Pu(VI) ion concentration. The detection limit of Pu(VI) was estimated to be 0.5 µg mL^-1 (3σ) in 3 M HNO₃. By the proposed method, Pu concentration was successfully determined in a nuclear waste solution for use in nuclear materials management.

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A selective pressurized liquid extraction procedure (SPLE) was developed for a fast determination of polychlorinated biphenyls in sediment. The final method was performed at 100°C with heptane/dichloromethane (90:10, v/v) as extraction solvent for 2 × 5 min. Sulfuric acid impregnated silica was placed downstream of the sample in the extraction cell to remove interfering components. This simultaneous extraction/clean-up was performed in 20 min, with an average congener recovery of 92% compared to a classical 24 h Soxhlet methodology and 2 h of external manual clean-up.

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On the basis of the structural interactions between diphenhydramine (DPH) and ephedrine (EPH) to enhance electrochemiluminescence (ECL) intensity, a flow-injection ECL analysis of DPH in the present of EPH by utilizing tris(2,2′-bipyridine)ruthenium(II), (Ru(bpy)₃²⁺) has been reported. In the optimized experimental conditions, the linear ECL response to concentrations of DPH is from 2.00 to 40.0 µg/L with a correlation coefficient of 0.9995 and a detection limit of 1.20 µg/L. The relative standard deviation (RSD) was less than 4.6% (n = 5) and the recovery was in the range of 98 - 106% for the determination of DPH in pharmaceutical samples. The method avoids any interference of coexistent EPH in the compounding drug without prior separation. The method has advantages over HPLC method in terms of speed and convenience, economics and safe procedure, and could be an alternative for places where HPLC equipment is not available.

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Although red phosphorus is used as a flame retardant for polymer materials, no analysis methods for it in resins has been established. Analysis methods for red phosphorus in resins were investigated using pyrolysis-gas chromatograpy/mass spectrometry (Py-GC/MS) by paying attention to the fact that it has a sublimation property. We found that the mass spectrum of red phosphorus shows a series of ions at m/z = 62, 93 and 124, and a fragment pattern indicating that red phosphorus (P₄ = 124, P = 31) was pyrolytically decomposed. The coefficient of the correlation between the content of red phosphorus in the resin and the peak intensity in Py-GC/MS was 0.9781. The relative standard deviations of this analysis method was 6.29% (n = 5). Py-GC/MS was applicable not only to qualitative analysis but also to the quantitative analysis of red phosphorus.

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Polyion complexes of three chitosans with poly(vinyl sulfate) (PVS) and dodecylbenzene sulfonate (DBS) were examined by a potentiometric study that was to separately measure the pH of sample solutions individually prepared. Apparent formation constants (K_i) of ion association between the protonated amines of chitosan and the sulfates of PVS or the sulfonates of DBS were determined. The effects of pH, coexistent salt concentration, and molecular weight on the values of K_i were investigated in order to reveal the properties of the complexation. The values of K_i for chitosan-PVS were quite larger than that for chitosan-DBS. The deducing effect of the coexistent salt was strong against chitosan-PVS, but was weak against chitosan-DBS. Thus, chitosan-PVS complexes possessed a strong electrostatic binding, and chitosan-DBS complexes included a hydrophobic interaction. For chitosan-PVS complexes the effect of the coexistent salt was weaker for a high molecular weight of chitosan than for a low molecular weight.

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The cell-immobilization technique is used for the physical and chemical fixation of cells onto a solid support in order to increase their stability and capacity of substrate uptake. However, there is no apparatus to observe the microbial community's structure inside cell immobilizing polymeric carrier. In order to satisfy the demand of monitoring for the microbial distribution inside the carrier, we developed an automatic phase contrast microscopic monitoring system capable of determining the microbial density and distribution inside a cell-entrapped carrier automatically.

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