BUNSEKI KAGAKU Abstracts

We have developed cell-based fluorescent indicators for biological substances that are secreted from living cells, including peptide hormones, neurotransmitters and cytokines. By introducing enzymatic amplification, we showed that the cell-based fluorescent indicators exhibit exceptional sensitivity. This novel approach allowed us to visualize the secretion of picomolar concentrations of biological substances from living cells. We describe herein recent advances in cell-based fluorescent indicators.

With recent progress in genetic engineering technologies, new fusion protein probes carrying genetically engineered photoproteins have been utilized for illuminating the bioactivity of a target substance. Recently, technologies for molecular imaging of a target substance in cell lines have been demonstrated in broad applications, such as the determination of bioactive small molecules, protein-protein interactions, and conformational changes of a target protein. In the present contribution, we introduce recent achievements in molecular imaging studies based on bioluminescence in our laboratory. This includes the molecular imaging of nuclear trafficking of transcription factors and nongenomic protein-protein interactions in the cytosol. In addition, we describe an integrated molecule-format bioluminescent probe, where all of the components for ligand sensing and light emission are integrated in a single molecule format. This probe was redesigned as a multicolor imaging measure for tracing multiple signal transduction pathways in response to exogenous stimulations. We also demonstrate new breakthroughs to improve ligand sensitivity through circular permutation and genetic manipulation of a small bioluminescent enzyme. We intend with this contribution that researchers in the field of analytical sciences will be able to recognize the joy of molecular imaging studies.

Many kinds of small-molecule organic chemical indicators have been developed that change their spectra or fluorescence intensity by binding to other small molecules or ions. Fluorescent probes equipping such molecular-switch systems are powerful tools for molecular imaging. The finding of fluorescent proteins enables us to develop genetically encoded fluorescent probes for visualizing molecules or cells in living animals and plants. We have attempted to create probes by equipping molecular switch systems using fluorescent proteins. In this review, we introduce three kinds of our approaches to develop and apply molecular switch systems to fluorescent protein-based probes.

Fluorescence bioimaging is one of the important technologies for biomedical applications to visualize phenomena in biological systems. Current problems of the bioimaging, such as color fading of phosphors, strong scattering and background illumination due to auto-fluorescence, are mostly caused by using short wavelengths for exciting fluorescence. To solve all of them, the authors have investigated an imaging system to use near-infrared light as an excitation source. For achieving this, rare-earth-doped ceramic nanophosphors are used to emit fluorescence efficiently. Collaboration concerning these technologies starting from the atomic scale, and reaching to the macroscopic scale through the nano scale is essential for the development of the system. In this review article, the development of a system using polyscale technologies is overviewed.

We have constructed a time-resolved fluorescence lifetime imaging (FLIM) system to perform quantitative observation of microenvironments and physiological parameters of a single cell. Fluorescence intensity depends on a variety of biophysical and experimental factors such as concentration or optical condition, whereas fluorescence lifetime is an inherent property of a chromophore, and is therefore independent of photobleaching, excitation power, and other factors that limit intensity measurements. FLIM techniques therefore provide detailed information on the environment in a cell. In the present study, we have used FLIM to examine stress-induced changes in a cellular microenvironment. We expressed the enhanced green fluorescent protein (EGFP)-fusion protein in HeLa cells and examined the time course of its fluorescence lifetime under cell stress. Cell stress was induced by the treatment with medium lacking nutrition and exposure to normal air. The cells have been found to exhibit a decrease in fluorescence lifetime with the cell stress. The observed decrease in the fluorescence lifetime has been interpreted in terms of a change in local electric field produced by the protein matrix surrounding the chromophore of EGFP. The fluorescence lifetime image of EGFP in HeLa cells has also been measured with varying intracellular pH. The pH dependence of the fluorescence lifetime could be measured using monensin that equalizes intracellular pH to extracellular one. It has been found that the fluorescence lifetime of EGFP decreases with decreasing intracellular pH after photoexcitation of its neutral chromophore, which can be explained by the pH-dependent ionic equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP. These results indicate that the intracellular pH of a single cell can be evaluated using FLIM of EGFP. From these results, we have proposed that FLIM can be used for noninvasive determination of the status of individual cells.

A microscope was combined with a picosecond dye laser and an intensified charge-coupled-device camera. The resulting fluorescence lifetime imaging microscope (FLIM) was applied to biological cells, e.g., cancer cells. The present technique has sufficient sensitivity, and allowed us to measure a wide FLIM image within a few minutes. Therefore, the FLIM system developed in this study can be widely used to explain a variety of biological phenomena in living cells.

A novel chemiluminescence analysis to taked advantage of a specific chemiluminescence reaction space around the liquid–liquid interface in a micro-channel under laminar flow conditions has been reported since 2004. We call the analytical system “micro-channel chemiluminescence analysis (MCCLA)”. First, the concept of micro-channel chemiluminescence analysis and its features were described and considered through direct observations of fluorescence and chemiluminescence using a fluorescence microscope-CCD camera and a microscope CCD camera. Next, several concrete examples of micro-channel chemiluminescence analysis were introduced, and the experimental data were basically discussed. A fluorescence compound was examined by micro-channel chemiluminescence analysis using an oxalate chemiluminescence reaction. Anti-oxygen, beverages, and saliva were also investigated by chemiluminescence analysis using a singlet oxygen chemiluminescence reaction. Furthermore, the copper(II) catalyst was determined by chemiluminescence analysis using a luminol reaction. In addition we briefly described feature planes concerning the micro-channel chemiluminescence analysis.

Time-resolved fluorescence spectroscopy using coumarin dyes as a fluorescent probe provides information on solvation dynamics in the vicinity of the coumarin dyes. In the present study, solvation dynamics occurred inside mesostructured silica with a uniform silica-mesopore diameter was examined by using four kinds of coumarin dyes as a fluorescent probe. In the silica-CTAB nanocomposite composed of CTAB (cetyltrimethylammonium bromide) micelles and silica-mesopores (pore diameter : 3.4 nm), it was found that coumarin dyes with different functional groups were located at different regions and that the molecular motions were differently constrained depending on the location of the coumarin dyes. In the silica-mesopores (pore diameter : 3.1 nm) filled with alcohol solvents, relatively small alcohols (ethanol and butanol) exhibited slow solvent relaxation process due to their confinment in the silica-mesopore, whereas such slow relaxation process was not recognized for large alcohols (hexanol and decanol).

Microchip electrophoresis (MCE) is one of the most suitable methods for biomolecular separation and analysis because of its superior characteristics. In biomolecular separation and analysis, laser induced fluorescent (LIF) detection is frequently employed in MCE and attains high sensitive detection. However, LIF detection requires the labeling of analytes, which contains troublesome procedures and results in decreasing of the separation efficiency. In addition, the labeling reaction at low concentration is significantly difficult, and thus true high sensitive detection is not easily attained and has been desired. Here, we report on the possibility of cup-stacked carbon nanotubes (CSCNTs) for high sensitive label-free detection and high separation efficiency. At first we investigated fluorescence properties of CSCNTs. Our investigation revealed that supernatant solution of CSCNTs after centrifugation had fluorescence at around 500 nm, while CSCNTs suspension did not. The application of CSCNTs for MCE enabled us to successfully separate and detect DNA without labeling it. Compared to the direct detection method, our method resulted in poor separation efficiency. However, by controlling of the CSCNTs’ aspect ratio and the immobilization of functional molecules on the CSCNTs surfaces, we could improve separation efficiency and attain the separation of various samples without labeling samples.

The use of a reversed micellar medium of cetyltrimethylammonium chloride (CTAC) in 1-hexanol–cyclohexane has been examined for the flow-injection (FI) determination of cobalt and manganese in organic solvents using the chemiluminescence (CL) reaction of luminol. The FI procedure used simply involves the mixing of a cobalt(II) acetate or manganese(II) acetate solution with luminol in a reversed micellar solution of CTAC in 1-hexanol–cyclohexane (1 : 25 v/v)/water (0.08 mol dm⁻³ sodium hydroxide). When mixed with a reversed micellar luminol reagent, either metal acetate caused a pronounced enhancement in the CL emission. An increase in the CL emission was produced by the addition of sodium carbonate to an aqueous phase containing luminol at a relatively lower sodium hydroxide concentration of 0.02 mol dm⁻³ dispersed in the reversed micellar solution. When the mole fraction of 1-hexanol in the bulk solvent was increased, a decline in the CL intensity was observed beyond a mole fraction of 0.042. For the cobalt and manganese determinations using 1-hexanol–cyclohexane as a solvent in the sample solutions, the optimum conditions were evaluated, and detection limits of 0.1 and 0.5 ng dm⁻³, respectively, were achieved. The calibration graphs obtained for cobalt and manganese were linear with dynamic ranges from 0.01 to 10 and 0.01 to 20 μg dm⁻³, respectively. The present FI-CL method was suitable for the determination of trace cobalt and manganese in various organic solvents.

Flow-injection analysis with chemiluminescence detection was investigated to determine small amounts of cobalt(II) ions in the presence of umbelliferon, hydrogen peroxide and cationic surfactants. The proposed flow system is comprised of three flow lines for cobalt (line 1), detection–reagents containing a surfactant (line 2) and hydrogen peroxide (line 3), respectively. The effects of cationic surfactant micelles on the chemiluminescence intensity are described. Among the examined cationic surfactant, cetyldimethylethylammonium bromide (CEAB) provided the highest enhancement on the CL intensity. Many kinds of radicals were created in this reaction system based on the addition of hydrogen peroxide. Furthermore, superoxide radical forming from hydrogen peroxide changed into a singlet state oxygen (¹O₂). The authors also investigated the effect of ¹O₂ scavengers on CL-enhancement. The CL intensity decreased to 0.1% using L-histidine and to 11.5% with hydroquinone as a ¹O₂ scavenger. As a result, the mechanism of CL-phenomenon was estimated to be the reaction between umbelliferon and ¹O₂.

The limit of determination of cobalt(II) by the proposed method was 1.2 ppb. The conditions for the determination of cobalt(II) were as follows : umbelliferone concentration, 1.5×10⁻³ mol/L (line 2, 0.8 mL/min); H₂O₂ concentration, 1.5% (line 3, 0.4 mL/min); pH in sample solution, 8 ; CEAB concentration, 8.0×10⁻³ mol/L ; reaction temperature, 25°C ; flow rate of sample solution (line 1), 0.8 mL/min.

A new method for the measurement of orthophosphate in land water has been developed. Molybdophosphate, formed between orthophosphate and molybdate in a sulphuric acid solution, was extracted into 4-methyl-2-pentanone with a tri-n-propylammonium ion as a counter-ion. After evaporation of the solvent, the residue was dissolved with acetonitrile. An aliquot of this solution was injected into a FIA system. Tri-n-propylamine was measured with Ru(bpy)₃^3＋ chemiluminescence detection. The calibration curve for standard phosphate was linear in the range 0.01〜10 μM, and the detection limit was 7.3 nM (S/N＝3). Orthophosphate in real samples, such as river and mineral water, was successfully determined using the present method.

Helium microwave-induced plasma (He-MIP) for fine-particle analysis was generated using 2.45 GHz microwave power (〜1 kW) with an Okamoto cavity of the surface wave mode at atmospheric pressure. The iron excitation temperature and the electron density of the plasma were measured. With an input microwave power of 800 W, an excitation temperature of the high-energy part of 8000 K, and an electron density of 2×10¹⁴/cm³ were obtained. The sample particles in an Al plate ablated by a Q-switched Nd : YAG laser (266 nm, 30 mW, 10 Hz, 3〜5 ns) or nebulization of suspension of Al₂O₃ powder (mean dia. 1 μm) was introduced into the center of an annular He-MIP through an inner tube of a torch along with the carrier gas (He or Ar). The time-resolved atomic emission spectrometry (AES) was studied for a particulate composition analysis of the laser ablation and the nebulization of suspension. Preliminary experimental data were obtained. The He-MIP AES could be detected with sufficient sensitivity.

The absolute fluorescence quantum yield of a solid sample was measured by using a fluorescence spectrophotometer equipped with an integrating sphere. However, there were difficulties in making a spectrum correction of the integrating sphere. To solve this problem, we developed a simple method for determining its wavelength characteristics, by using the ratio of the diffuser to the integrating sphere. They were measured by combining both scan data with corrected excitation light. As a result, we were able to measure the corrected spectrum over a wide wavelength range of 200 nm to 800 nm. The experimental value of the fluorescence quantum yield of MgWO₄ was 0.80 (RSD 0.6%) with the present method, which is a good agreement with a reported value (0.81).

Recently, environmental pollution by toxic metals, such as Cd, Pb, Cr, and so on, is getting increasingly worse. In order to increase measurement chances, a portable elemental analyzer is expected to be developed. Conventionally, the measurement of trace metals in environmental pollution was done by atomic absorption spectrometry (AAS), or inductively coupled plasma atomic emission spectrometry (ICP-AES). However those instruments are very expensive, and require a large amount of Ar gas, a nebulizer, and a high power source, and is thus not suitable for portable use. Liquid electrode plasma atomic emission spectrometry (LEP-AES) is a novel atomic emission spectrometry, where a sample solution is put into micro channel whose center is made to be narrower. A high voltage is applied to the solution from both ends, and then a micro-plasma is generated in the channel. This technique is different from ICP-AES due to the following points : it doesn’t require a plasma gas or a nebulizer, and an ultra compact and portable element analyzer can be made. In this study, the performance of this analyzer was investigated with a solid phase extraction (SPE) method to measure the Pb concentration in soil. At first, the basic performance was confirmed without SPE. In the result, it is found that the limits of detection (LOD) for Pb by this system is 1.3 mg/L at 405.8 nm, the relative standard deviation is under 10%, and the calibration curve has good linearity at a Pb concentration of less than 800 mg/L. Those values are good enough to measure Pb in soil. Next, heavy metals in standard soil sample (sea bottom material) were extracted into water by the official method. Lead in the extract was separated from other elements by solid-phase extraction, and was then concentrated. By measuring the SPE elution using the LEP-AES system, the original concentration of Pb in the soil was determined to be 80.2 mg/kg (dry), which was close to the certificated value of the soil of 82.7±3.8 mg/kg.

The laser-induced fluorescence of asbestos (chrysotile, crocidolite, and amosite) and several types of building materials such as glass-wool and talc, has been studied in an attempt to discriminate asbestos from other materials. The fluorescence spectra induced by ultra-violet (266 nm) laser pulses were observed over the wavelength range from 350 to 700 nm, and differences in the shape of the spectrum between asbestos and other materials was identified. The lifetimes and total fluorescence intensity in the visible region were also investigated and evaluated for the numerical discrimination of asbestos. In particular, significant differences in the shape and the total fluorescence intensity were observed between asbestos and fibrous building materials. The total fluorescence intensities of these materials were more than tens-times as large as those of asbestos. Several methods used to discriminate asbestos by the differences in the fluorescence characteristics are proposed.