BUNSEKI KAGAKU Abstracts

Microchip-based immunoassay, which is immunoassay performed on a microchip, has recently been used in various fields owing to its advantages, such as reduction in sample and reagent consumption, short analysis time and simple operation. Different types of immunoassay have been applied to the miniaturization on a microchip. Material and surface modifications of a microchip, introducing and moving samples and detection must be taken into consideration in order to realize a microchip-based immunoassay. Also, the microchip system should be designed according to a type of immunoassay. In the present review article, we explain the types and classifications of immunoassays and describe important points for developing a microchip-based immunoassay. We then introduce several examples of microchip-based immunoassay.

We have developed a metal tube atomizer for the electrothermal atomization atomic absorption spectrometry (ETA-AAS). Tungsten, molybdenum, platinum tube atomizers were used as the metal atomizer for ETA-AAS. The atomization characteristics of various metals using these metal tube atomizers were investigated. The effects of heating rate of atomizer, atomization temperature, pyrolysis temperature, argon purge gas flow rate and hydrogen addition on the atomic absorption signal were investigated for the evaluation of atomization characteristics. Moreover, ETA-AAS with metal tube atomizer has been combined with the slurry-sampling techniques. Ultrasonic slurry-sampling ETA-AAS with metal tube atomizer were effective for the determination of trace metal elements in biological materials, calcium drug samples, herbal medicine samples, vegetable samples and fish samples. Furthermore, a preconcentration method of trace metals involving adsorption on a metal wire has been applied to ETA-AAS with metal tube atomizer.

The present paper describes a voltammetric study on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion-transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By the addition of a small amount of hydrophobic ion into W1 or/and W2 containing a hydrophilic salt serving as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with a counter ion to hold the electroneutrality within the BLM phase. It was found that the counter ion transferred between W1 and W2 across the BLM upon applying a membrane potential. As for the facilitated transport of alkali ions across a BLM containing Val, which served as an ion carrier compound, it could be interpreted by considering not only the formation of an alkali metal ion-Val complex, but also the distribution of both the objective cation and the counter ion. In the case of the addition of GA as a channel-forming compound into the BLM, the facilitated transports of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transports of alkali ions across the BLM could be explained by considering the hydrophobicity and the ionic radius of the counter ion.

Molybdenum blue method, widely used for the determination of phosphate, is based on the formation and reduction of 12-phosphomolybdic acid, in which ascorbic acid with antimony(III) and tin(II) chloride function as a reductant. In this study, various metal ions with ascorbic acid as a reductant were examined to form phosphomolybdenum blue. When bismuth(III) ion with ascorbic acid was used, the phosphomolybdenum blue(γ_max, 720 nm) was formed at room temperature. The amounts of bismuth in the phosphomolybdenum blue were determined by AAS, and the bismuth to phosphate ratios was found to be ca.2. Also, a new method using ascorbic acid with bismuth(III) was developed. The proposed procedure is as follows. An adequate amount of sample is taken in a 50 mL measuring flask and diluted to about 40 mL with water. A 5 mL of sulfuric acid(1.73 M)-ammoniummolybdate (8.3 g/L)-bismuth nitrate(0.1 g/L as Bi) solution and 1 mL of L-ascorbic acid (50 g/L) solution are added and diluted to 50 mL with water. After standing for 10 min at room temperature, the absorbance is measured at 720 nm. By the proposed method, a liner relationship was obtained between the absorbance and the concentration of phosphate in the range of 0.01〜0.15 mg/50 mL. Good results were obtained in recovery tests using river water containing phosphate in the range of 0.01〜0.15 mg.

Coloration of protein employing the reaction of a tryptophan residue and an aldehyde was investigated. It was observed that the reaction of lysozyme with 4-(N,N-dimethylamino)benzaldehyde resulted in formation of a bluish product due to formation of a chromophore group. The coloring depends on chemical structure of aldehydes. Electrophoresis of colored lysozyme using a polyacrylamide gel showed molecular weight bands corresponding to a monomer, a dimmer, and a trimer. The production of an oligomer was also confirmed by MALDI TOF-MS spectroscopy. Further, it is suggested that the present procedure is applicable to transcription of protein on a membrane by a western blotting technique.

Trimethylamine (TMA) is an unpleasant odor compound, like ammonia. Conventional gas chromatography with a flame ionization detector (FID) was not suitable for the determination of trace TMA in air, because of troublesome concentration procedures and the presence of interfering organic substances. In this study, we developed a detection system method for trace TMA in ambient air by using a headspace gas chromatography-surface ionization detector (SID). An air sample was trapped in a diluted a phosphate solution by bubbler sampling. Then, a potasium hydroxide solution (50 wt％) was added to a sample solution in a head space vial to volatilize TMA. After heating the vial at 30°C for 5 minutes, the head space gas was injected to GC/SID by a gastight syringe. The method was applied to the determination of TMA in the air in a public lavatory. When FID was used to analyze, TMA was not separated from other organic substances. In this method, trace TMA was succesfully detected without interferences from other substances, because of excellent sensitivity and selectivity of SID to tertiary amines. The detection limit was 0.09 ppbv for 120 L of an air sample.

A target value for diquat residues in tap water is set at 5 μg/L in Japan. We succeeded in the quantitative analysis of diquat in water samples at 1/100 of the target value by hydrophilic interaction chromatography/electrospray ionization/mass spectrometry (HILIC/ESI/MS). For solid-phase extraction we used a new mixed-mode cartridge comprising a weak cation exchange and reversed-phase retention. A 50 mL of water sample spiked diquat-d₄ was passed through the cartridge. Diquat was eluted with 4 mL of acetnitorile and 2-propanol (60 : 40) containing 4％ trifluoroacetic acid. The elute was evaporated to dryness at 40°C under nitrogen gas. The test sample was dissolved in 100 μL of the mobile phase for analysis by HILIC/ESI/MS. The mobile phase consisted of acetonitrile and 10 mM ammonium formate (pH 3.7)＝50 : 50. In the ESI positive-ion mode, m/z 183, m/z 184, and m/z 157 were measured for diquat, and m/z 186 for diquat-d₄. The recovery rates at concentrations of 0.05, 0.5, and 5 μg/L in distilled water, tap water, and river water were 88〜105％, and the CV values were 1.7〜15％. The concentrations of diquat in water were determined using the proposed method. These were 12 tap water samples and 30 raw water samples (river, 14; lake, 5; shallow well, 9; deep well, 2) collected from Hyogo prefecture. As a result, diquat was detected in one raw water sample (shallow well, 2.8 μg/L).

Inductively coupled plasma-optical emission spectrometry (ICP-OES) was applied for the determination of sulfur in biomass ethanol and compared with three conventional combustion-based methods of microcoulometry, ultraviolet fluorescence and oxyhydrogen flame combustion. The three combustion-based methods gave similar results. However, ICP-OES with standard solutions prepared from sulfate form-sulfur caused a serious overestimate due to the presence of dimethyl sulfide (DMS). The emission intensity of DMS form-sulfur was much higher than that of sulfate form-sulfur due to its high introducing efficiency to a plasma. Consequently, DMS form-sulfur caused a 6 to 21-times overestimate, depending on the ICP instruments used. In order to suppress this phenomenon, it was recommended to carry out PTFE closed-vessel acid decomposition with a mixture of hydrogen peroxide and acetic acid prior to an ICP-OES measurement.

The concentration of cyanogen chloride (CNCl) in the Yokohama aqueduct was higher than the standard of Japanese water quality. In the present study, we investigated what produces CNCl in water. Our results showed that CNCl was generated in large quantities when water containing chloramines was buffered with tartaric acid. On the other hand, when it was buffered with some other buffer solution, such as acetic acid, phosphoric acid, phthalic acid or sulfuric acid, CNCl was hardly generated. Therefore, a tartaric acid buffer solution seemed to cause the generation of CNCl.