The nano-kinetics movement of a single DNA molecule was visualized by a newly developed video-microscope system with an optical fiber, called a “pin-fiber video scope”. The stretching and shrinking motion was clearly observed, and its kinetics was analyzed by numerical calculations. The new video-microscope system has a potential to analyze the nano-kinetics of a molecule.
In this research, we synthesized a novel DNA-polymer conjugate and evaluated its application to an affinity precipitation separation of TATA-box binding protein (TBP), which is a representative general transcription factor. The conjugate was composed of two fractions. One was a double-stranded DNA modified by the grafting of poly(N-isopropylacrylamide) (PNIPAAm), which is known as a thermosensitive vinyl polymer. The other fraction is a native double-stranded DNA containing a specific base sequence (5′-TATAAA-3′) called a TATA-box. These two fractions, which have EcoRI termini, were treated with T4 DNA ligase, and the block conjugate was obtained as a precipitate after two wash processes. When the resultant block conjugate was introduced into a sample solution containing TBP (0.26 μM) and bovine serum albumin (BSA) (0.39 μM), a rapid and selective precipitation separation of TBP under homogeneous conditions was achieved by controlling temperature. The purity of TBP in the precipitation fraction was estimated to be above 90%.
Derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) has been examined. Under APCI conditions, gas-phase electrons are provided by the corona discharge and captured by electron-affinitive compounds. In negative APCI-MS, therefore, ultrahigh sensitivity can be obtained by tagging neutral steroids, whose ionization efficiencies are low in the conventional APCI-MS, with electron-capturing moieties, such as a nitro group. We synthesized various boronic acid and hydrazine derivatives having electron-capturing moieties as derivatization reagents for 1,2-diol compounds and oxosteroids, respectively. Among reagents examined, those having the 2-nitro-4-trifluoromethylphenyl moiety were most effective in increasing sensitivity. That is, the detection responses of the derivatives with these reagents were increased by several to more than 200-fold over intact steroids, where limits of detection were some picograms. The developed derivatization procedures were applied to analyses of small amounts of steroids in human plasma and gave satisfactory results.
Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype γ1, κ) showed an affinity constant (Ka) for cortisol of 1 × 109 M-1 and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17α-hydroxyprogesterone (12%), 6β-hydroxycortisol (8.4%), and tetrahydrocortisol (<0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 ± 17.0 µg/day (n = 7) and 80.8 ± 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.
The quantification of glucose by using a multi-channel dissolved oxygen (DO) meter (DOX96) with immobilized glucose oxidase (GOD) and mutarotase (MUT) was performed. An evaluation of the inhibitory activities for α-glucosidase (AGH) by modifying our batch-type pseudo-in vivo assay system [Oki et al.; Biol. Pharm. Bull., 2000, 232, 1084] was also performed using a DOX96. When 45 U/well GOD and 18.75 U/well MUT were immobilized on the surface of a gelatin membrane on the electrodes, the response shown by the decrease percent of DO (%) obtained with 8 electrode wells in the same row was linear with the glucose concentration up to 3.3 mM and a correlation coefficient larger than 0.9. To estimate the AGH inhibitory activity, AGH-immobilized Sepharose supports in the well of a silent screen plate were used. The IC50 values of acarbose and 1-deoxynojirimycin, a medicinal inhibitor for diabetes, were 0.70 ± 0.08 µM and 0.40 ± 0.13 µM, respectively, and coincided well with those by a pseudo-in vivo assay.
In order to obtain the full spectrum from 400 to 800 nm of each pixcel of a microscopic image, a unique spectro-imaging system was developed using an image slicer. The image slicer is composed of 100 photo fibers which are arranged in a matrix of 10 × 10 at the entrance and 100 × 1 at the exit. A line of this 100 signals is passed through a glism and projected onto a CCD. This system was applied to the fluorescent imaging of bio-cells. One of the demonstrative examples was simultaneous measurements of the Ca2+ concentration and the pH using of respective fluorescent probes. An electric signal was applied to BY-2 protoplasts and the fluorescent spectrum from 500 nm to 800 nm was measured every 5 s. The spectrum of the BY-2 protoplasts changed in response to the electric signal and the Ca2+ concentration, and the pH changes could be monitored. The wavelength resolution was satisfactory, but the space resolution was still rough in comparison with the usual microscopic systems. Notwithstanding these conditions, we could obtain discrete data from more than several tens of sites in a single-cell or a chain of several cells.
The continuous monitoring of the distribution of glutamate (Glu), a neurotransmitter released at synaptic terminals, is important in terms of understanding the signal transfer mechanism in the brain. In this study, we monitored the concentration of Glu released at multiple positions in a hippocampal slice continuously, and obtained an approximate Glu distribution by using our electrochemical glutamate sensor array. After confirming our sensor’ s high sensitivity to Glu, we placed a slice on the array, and measured the currents at selected electrodes in the array. When we stimulated a specific position in the slice electrically, the glutamate concentration increased in different areas after several tens of seconds. The presence of glutamate receptor blockers suppressed these increases. This suggests that the electrical signal was transferred along with neurons through synapses and stimulated the Glu release. Our multichannel glutamate sensor should be a powerful tool to determining the distribution of real-time glutamate non-invasively for the studies using biological samples.
The coherent detection imaging (CDI) method the optical heterodyne detection technique. CW and single frequency lasers having long coherence lengths are used to exploit the maximum advantages of heterodyne detection, such as high directionality, selectivity and sensitivity. The CDI method based on optical heterodyne detection enables selective filtering of the directional coherence-retaining emergent photons, which leads to image reconstruction from projections, similar to X-ray computed tomography (CT). So far we have demonstrated the advantages and capabilities of the measurement technique for transillumi-nation optical computed tomography in biomedicine. Here, we investigate the fundamental imaging properties of CDI method, such as its high directionality and quantitativeness, with preliminary physical phantom experiments. The results show that the CDI method satisfies the requirements for CT reconstruction under the first order approximation, and enables quantitative measurements in the sense that the relationship between estimated and actual concentration retains a satisfactory linearity.
A novel PVC membrane electrode for the determination of scopolamine ion based on the formation of an ion-association complex of scopolamine with the phosphotungstate counter anion as an electroactive material dispersed in a PVC matrix is described. The sensor shows a fast, stable, near-Nernstian response for 1 × 10-2 mol dm-3 to 1 × 10-6 mol dm-3 scopolamine at 25°C over the pH range of 3 − 7 with a cationic slope of 54.5 ± 0.5 mV/decade. The lower detection limit is 8 × 10-7 mol dm-3 and the response time is 15 − 45 s. The selectivity coefficients for scopolamine relative to the number of interfering substances were investigated. There was negligible interference from the studied cations, anions, and pharmaceutical excipients. The determination of scopolamine in aqueous solution shows an average recovery of 100.0% and a mean relative standard deviation (RSD) of 1.5% at 500 µg/cm3. The direct determination of scopolamine in some formulations (scopolamine injection and eye drops) gave results that compare favorably with those obtained by the United State of Pharmacopoeia method. Potentiometric titration of scopolamine with sodium tetraphenylborate and phosphotungstic acid as a titrant was monitored with the developed scopolamine electrode as an end point indicator electrode.
A monolayer of Nile Blue (NB) has been covalently immobilized on the self-assembled thiol-monolayer modified gold electrode. Cyclic voltammograms indicated a stable and reverse redox process of NB bonded on the electrode surface. The mechanisms of redox process coupling with proton transfer were proposed. The NB-modified electrode showed excellent electrocatalytic activity toward Nicotinamide adenine dinucleotide (NADH) oxidation and horseradish peroxidase (HRP) reduction. A hydrogen peroxide biosensor based on NB as a mediator has been demonstrated.
The adsorption characteristics of As(V) and As(III) on titanium dioxide loaded Amberlite XAD-7 resin have been studied. The resin was prepared by impregnation of Ti(OC2H5)4 followed by hydrolysis with ammonium hydroxide. Batch adsorption experiments were carried out as a function of the pH, shaking time and the concentration of As(V) and As(III) ions. The resin showed a strong adsorption for As(V) from pH 1 to 5 and for As(III) from pH 5 to 10. The adsorption isotherm data for As(V) at pH 4 fitted well to a Langmuir equation with a binding constant of 59 dm3 mol-1 and a capacity constant of 0.063 mmol g-1. The data for As(III) at pH 7 also fitted well to a Langmuir equation with a binding constant of 5.4 dm3 mol-1 and a capacity constant of 0.13 mmol g-1. The effect of diverse ions on the adsorption of arsenic was also studied. Column adsorption experiments showed that the adsorption of As(III) is more favorable compared to As(V), due to both the faster adsorption and larger capacity for As(III) than As(V).
Structural control of Schiff base ligands for selective extraction of copper(II) was investigated by changing pendant arms and the distance between two imine-N donor atoms in ligands. Di-Schiff base ligands, N,N′-bis(2-quinolylmethylidene)-1,2- diiminoethane (BQIE), N,N′-bis(2-pyridylmethylidene)-1,3-diimino-2,2-dimethylpropane (BPMP) and N,N′-bis(2- quinolylmethylidene)-1,3-diimino-2,2-dimethylpropane (BQMP), were used as complexation reagents for ion-pair extraction of divalent transition metal cations into nitrobenzene with picrate anion. The pendant arms affected the lipophilicity of ligand to nitrobenzene, due to their polarity. The distance between two imine-N atoms, on the contrary, was a factor of controlling the extraction selectivity. BQMP has both 2-quinolyl pendant arms and trimethylene backbone structure; use of BQMP as a complexation reagent led to the selective extraction of Cu2+ in the system.
Poly(ethylene glycol) (PEG) in water is known to alter the structure and/or state of water to give a different polarity from that of pure water. We determined using the solvatochromic comparison method the dipolarity/polarizability (π*), hydrogen bond (HB) accepting basicity (β) and HB donating acidity (α) of aqueous solutions of PEGs of variegated molecular weights at different concentrations in order to understand the influence of the polymer on these properties of water. It was observed that PEG decreases α for water while it does not change π* and β appreciably in the range of the molecular weight and compositions studied.
Oxidation of Cr(III) during sonication in carbonated aqueous solutions saturated with CCl4 leads to the quantitative formation of Cr(VI) and provides a simple and rapid method for spectrophotometric chromium determination with 1,5-diphenylcarbazide. The key to this method is the production of chlorine radicals when aqueous solution saturated with CCl4 is exposed to ultrasonic waves of 40 kHz. The effects of sonication period, CCl4 solution volume, acidity, and interferences were discussed. The time required for a single determination is lower than 2 min. The relative standard deviation obtained for aqueous solutions with 1 µg of Cr was <2% (N = 10) and the calculated detection limit (3σ) was 5 ng of Cr.
A microscopic photothermal lensing measurement under two-color continuous-wave laser excitation was performed to investigate a signal enhancement owing to the transient absorption by photoexcited solute molecules in liquid solutions. An intensity-modulated 409 nm laser beam and an un-modulated 532 nm laser beam were used for excitation, and a 670 nm probe beam was used for detecting the modulation amplitude of thermal lensing signals generated with a microscopic objective lens focusing laser beams into a capillary flow cell of 0.1 mm optical path length. The amplitude of the modulated signal increased as the power of the un-modulated laser beam increased, and a 143-times magnification was observed for an iso-propanol solution of naphthacene having 4.6 × 10-4 absorbance at 409 nm and a negligible absorbance at 532 nm. A four-level model explaining the signal enhancement is proposed, and an important role of the transient absorption by photoexcited molecules is discussed.