The technique of Hadamard transform microscopic fluorescence imaging is described. With an Acridine Orange (AO) staining cell, some factors that influence the fluorescence intensity of a cellular AO-DNA complex were studied; thereby the technique was applied to measure the nuclear DNA content (ploidy) in a breast tumor cell, and some parameters were employed to evaluate the tumor malignancy. A comparative study with conventional microfluorometry indicates that our system has some outstanding advantages. By using this system, the results of malignancy evaluation for 38 cases of breast tumor specimens were concordant with pathological diagnosis. This work demonstrates that this technique has a high potential value in medicine and cytobiology and may be applied as a new method for diagnostic and prognostic studies of tumor cases.
A tetradentate β-diketonate europium chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chloro-sulfo-o-terphenyl(BHHCT)-Eu3+, was labeled to a streptavidin-bovine serum albumin conjugate for time-resolved fluorometric detection of λDNA by hybridization with a biotinylated probe DNA in a microtiter well. The method gave a detection limit of 23 pg/well (solid-phase measurement) and 4 pg/well (solution-phase measurement) for specific DNA.
A tetradentate β-diketonate europium fluorescent chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu3+, was used as a label for highly sensitive time-resolved fluoroimmunoassay of bensulfuron-methyl (BSM). A BSM-BSA (BSA=bovine serum albumin) conjugate was labeled with BHHCT-Eu3+ and the labeled BSM-BSA conjugate was used for competitive immunoassay. The assay was carried out in a 96-well microtiter plate coated with the anti-BSM monoclonal antibody. The method gives the detection limit of 24 pg/ml, which is one to two orders of magnitude lower than those of the conventional HPLC and other methods. The RSD of the present method is less than 10%, and the recovery is in the range of 90 - 110% for BSM water samples.
Nitrite and nitrate in biological samples can act as an indicator of nitric oxide generated in vivo and have attracted a lot of attention as a messenger of diverse physiological processes. For the determination of such nitrogen oxides, a simple and sensitive flow-injection method coupled with a successful pretreatment method was developed. Nitrite and nitrate in sample solution were injected into a carrier solution containing EDTA and ammonium buffer, and flowed into the copperized cadmium (Cd/Cu) reduction column installed on-line. Here the nitrate was reduced to nitrite, and then nitrite was determined spectrophotometrically on the basis of a diazotization/coupling reaction. The detection limits for nitrite and nitrate were 5×10-8 M, and the sampling rate was about 40 samples per hour for nitrate determination. The biological samples, such as serum, plasma and cell culture medium, were deproteinized using a batchwise NaOH-ZnSO4 method before FIA measurement. The NaOH-ZnSO4 deproteinization method was very effective in term of the recovery of nitrate and the maintenance of the reduction efficiency of the Cd/Cu column. The relative standard deviations were 2.1 - 3.1%, and the recoveries were 95 - 100% for the determination of nitrite and nitrate in serum samples through the whole procedure. In the case of cell culture medium, the proposed deproteinization was especially effective for removing the interfering amino acids containing sulfur atom by using the reaction between Zn2+ in deproteinization agent and sulfur atom in analytical substances.
NAD(P)H coenzyme concentrations were determined with the combination of a nitrite-selective membrane electrode and a nitrate reductase enzyme. The method was based on the detection of nitrite formed by the enzymatic reaction of NAD(P)H with nitrate catalyzed by nitrate reductase. The lower detection limits for both these coenzymes were around 100 µM. This system was applied to detect glucose by combining an additional glucose dehydrogenase enzyme. Serum glucose levels could be determined through the detection of nitrite.
In order to make a molecule imprinting polymer (MIP) with highly chiral selectivity against N-t-Boc-L-Trp, a new kind of “cocktail” functional monomer: acrylamide+2-vinylpyridine was investigated. The MIP showed impressive chiral selectivity (α=3.23). With the increasing of water content in the mobile phase, ionic and hydrophobic interaction were found to be responsible for the chiral recognition process instead of the hydrogen bond. Tailing and peak asymmetry problems were overcome by using linear gradient elution. Physical properties such as thermal stability and pore structure for the MIP were also investigated.
Vanadium, chromium and molybdenum were determined by atomic absorption spectrometry with a coated graphite furnace. Pyrolytic graphite tubes were coated with boron carbide. A mixture of nitrates of nickel and strontium and boric acid was then added to a sample solution as a matrix modifier. Using this coated tube, the determinations of vanadium, chromium and molybdenum were successful with high sensitivity and better reproducibility. No interference from metal nitrates and chlorides with vanadium and molybdenum determinations and from metal nitrates with chromium determination was observed. The chloride interference with chromium and the sulfate interference with analyte could be removed by adding ammonium salt of EDTA.
A highly sensitive solid-phase spectrophotometric method for the determination of trace amounts of copper(II) using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid (TPPS) is described. The copper(II)-TPPS complex was extracted on a membrane filter from an acidic solution containing Zephiramine (benzyldimethyltetradecylammonium chloride) and a large excess of perchlorate ion, leaving excess TPPS in the solution. The solid-phase absorbance of the copper(II)-TPPS complex on the membrane filter was measured at 417 nm. A linear calibration graph was obtained for up to 20 ng of copper(II) in a 20 ml sample volume; the limit of detection was 0.15 ng, based on three-times the standard deviation of the blank value. The proposed method was applied to the determination of copper in tap, river and sea water samples. The results are in fair agreement with those obtained by suspension-introduction graphite-furnace atomic absorption spectrometry using ammonium pyrrolidinedithiocarbamate and finely divided anion-exchange resin.
For investigating the Belousov-Zhabotinsky (BZ) reactions in free-fall experiments under microgravity, conducted at Japan Microgravity Center (JAMIC) in Hokkaido, the automated setup of batch-type, which was constructed in this work, had to be placed in the limited space and to have functions to prepare the reaction solution; it was also required to send it to the vessels after the necessary induction period and to collect image data via CCD cameras, including the process of integrating the capsule. These operation sequences of each device involved simple solid state relays. The initial start of the relay sequences was performed by a start command from the operation room of JAMIC. The propagation profile of the chemical patterns under microgravity of 10-5 g was collected as image data for ca. 9 s. They were not disturbed by the release of carbon dioxide, which often deforms the chemical patterns in the aging of BZ reactions.
An alternative analytical procedure for cobalt determination by tungsten coil electrothermal atomic absorption spectrophotometry (TCAAS) was developed to determine the liquid ruminal passage rate (turnover) of Co-EDTA in sheep feces. A matrix-matching procedure and a selective extraction of Co in 1.0 mol l-1 HCl were evaluated in order to correct and minimize the interference effects caused by sample matrices. As application, six sheep received at the same time one dose of the marker (Co-EDTA); thier fecal samples were collected in intervals of 6 h during 90 h. The Co amounts determined in the dry sheep feces by TCAAS were compared with those obtained by a flow injection catalytic spectrophotometric method. The characteristic mass and the detection limit, both based on peak height absorbance, were 23.1 and 19.1 pg, respectively, for 10 ml of sample volume in the samples of sheep feces. The rsd was 0.5% for 10 consecutive injections of 20.0 mg Co l-1. The accuracy was assessed by employing the paired t-test at 95% confidence level; there was no significant difference for Co content determined by TCAAS and by flow injection spectrophotometry.