A novel cell-adhesion surface, controlled by nanometer-scale topography and chemical patterning, was developed using semiconductor fabrication methods and the formation of self-assembled monolayers. The patterned surface had a sharp contrast between the adsorption and non-adsorption of proteins and cells, and the contrast could be maintained for more than 10 days. The patterning method could easily realize a single cell array and control of the cell morphology. The nanometer-scale patterned surface could control cell adhesion and proliferation. Using the patterned surface will contribute to studies about cell-surface interactions.
The fluorescence of 4-(4′-dimethylamino)styryl-1-methylpyridinium (C1SP) was enhanced by more than 33-fold by complexation with β-cyclodextrin (β-CD) bearing a naphthalene, pyrene, or phenylboronic acid group. The great enhancement of the fluorescence of C1SP was due to a π-π stacking interaction, by which the bond rotation of C1SP was effectively suppressed. The results indicate that C1SP and structurally related hemicyanine dyes potentially become powerful fluorescent indicators for aromatic compounds through the π-π stacking interaction in water.
This review first describes the invention of functional interfaces to promote biochemical redox reactions between substrates in dipolar aprotic solvents and enzymes or related compounds immobilized at the interface. The interfaces contain hydrophilic polymer membranes, a gold nanoparticle self-assembled electrode constructed by using rigid rod dithiols, and binary self-assembled monolayers composed of amino and carboxyl terminal groups. Other topics covered are: the electrochemical characterization of the hydrophilic polymer membrane; the development of biosensors to obtain reaction parameters of enzymatic and of electrochemical kinetics; and applications to the study of materials involved in metabolism.
Cells are frequently exploited as processing components for integrated chemical systems, such as biochemical reactors and bioassay systems. By culturing vascular endothelial cells (ECs) in integrated chemical devices, vascular models have also been fabricated. Here, we utilized a thermally fused-glass microchip which is chemically and physically stable and favorable for optical detections, and cultured human arterial ECs (HAECs) in it. HAECs reached confluence within 4 days. Survival and tolerance for high shear stress (25 dyn/cm2) of the HAECs were confirmed. Furthermore, HAECs responded to inflammatory cytokine, tumor necrosis facor-α (TNF-α) and attached to more leukocyte cell line, HL-60 cells than unstimulated HAECs. Our developed device can be applied as a human arterial model, and we propose it as a new method for vascular studies.
A tetrakis-acridinyl peptide (TAP) cassette, consisting of a double-stranded region of alternating AT sequence bound to TAP and a single stranded overhanging sequence of continuous dA, was prepared by mixing TAP with d[A18(TA)51]. A TAP cassette could be applied to the fluorometric detection of hybridized DNA on the DNA chip, which was prepared by stamping a 45-meric DNA probe onto a gold-coated plastic chip using a high-precision spotter developed at RIKEN. Spots on the DNA chip were imaged by a CCD camera after hybridization with 65-meric target single-stranded DNAs carrying a continuous dA20 sequence (dA tail) on the DNA chip after treatment with a TAP cassette. Their fluorescence intensity on the DNA chip showed a good linear correlation with the concentration of the target DNAs in the range from 10 pM to 1 nM. Fluorescence of their spots derived from the TAP cassette remaining on the surface of the DNA chip through the dA tail of the hybridized target DNA. Furthermore, the TAP cassette could be successfully applied to the quantitative detection of complementary RNAs (cRNAs) prepared from rat brain with reverse transcription and in vitro transcription.
DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.
In this paper, we describe an amperometric-type enzymeless glucose sensing system based on a nanoporous platinum (Pt) electrode embedded in a microfluidic chip. This microchip system is comprised of a microfluidic transport channel network and a miniaturized electrochemical cell for nonenzymatic glucose sensing. Sample and buffer solutions were transferred to the cell by programmed electroosmotic flow (EOF). A nanoporous Pt electrode with the roughness factor of 200.6 was utilized to determine glucose concentrations in phosphate buffered saline (PBS) by the direct oxidation of glucose, without any separation process. The sensitivity of the developed system is 1.65 µA cm-2 mM-1 in the glucose concentration range from 1 - 10 mM in PBS.
A highly sensitive and simple electroanalytical methodology is presented using an in-situ bismuth film modified edge plane pyrolytic graphite electrode (BiF-EPPGE) which is exemplified with the simultaneous determination of cadmium(II) and lead(II). Square-wave anodic stripping voltammetry is utilised with the effects of several experimental variables studied. Simultaneous additions of cadmium(II) and lead(II) were investigated where two linear ranges between 0.1 - 100 and 0.1 - 300 µg/L and also detection limits of 0.062 and 0.084 µg/L were obtained, respectively. The method was then successfully applied to the simultaneous determination of cadmium(II) and lead(II) in spiked river water, where recoveries of 100.5 and 98% were obtained, respectively. This electroanalytical protocol using edge plane pyrolytic graphite electrodes is one of the simplest methodologies to date using non-mercury based electrodes and is simpler and cheaper than alternatives such as carbon nanotube electrode arrays, suggesting the use of edge plane pyrolytic graphite electrode for routine sensing.
A PVC membrane sensor for the selective determination of mebendazole (MBZ) was fabricated. The sensor is based on an ion association of MBZ with silicotungstic acid (STA) as ion pair and bis(2-ethylhexyl)phthalate (BEP) as the plasticizing agent in a PVC matrix. The sensor showed a linear response for MBZ for a concentration range 1.0 × 10-6 - 5.0 × 10-2 M with a Nernstian slope of 55.8 mV/decade (limit of detection 6.3 × 10-7 M) in the pH range 4 - 7. It has a fast response time of <30 s. The sensor showed a very good selectivity for MBZ with respect to a large number of ions. The direct determination of MBZ in pharmaceutical formulations gave results that compare well with the data obtained from the standard method.
An effective method for separating and enriching organophosphorous pesticides (OPPs) from vegetables by solvent sublation and the OPPs determination by gas chromatography with flame photometric detector (GC-FPD) has been developed. The effects of organic solvent, nitrogen flow rate, pH of the solution, sublation time etc. on the sublation efficiency of OPPs were investigated in detail, and the optimal conditions of solvent sublation were selected. The floated product of vegetables in the optimal conditions was determined by GC-FPD. The limits of detection (LODs) ranged from 1.2 µg kg-1 (for dimethoate) to 3.5 µg kg-1 (for chlorpyrifos). The recoveries of spiked vegetable samples were from 81.3 to 98.9%, and RSD values were from 0.46 to 4.83%. The results show that this method is simple, sensitive and rapid.
An isotope dilution liquid chromatography/tandem mass spectrometry is proposed as a reference method to determine the level of tyrosine in human serum. The advantages of this method include simple sample preparation without derivatization, the selective detection of compounds of interest in complex matrices, and the use of an isotopically labeled analogue as an internal standard. Tyrosine and its isotopically labeled analogue were monitored at a transfer m/z of 182.1/136.2 and 188.1/142.2, corresponding to [M+H]+/[M+H-HCOOH]+ in a multiple reaction monitoring mode. The expanded uncertainty for the measurement of tyrosine in the serum was approximately 0.95% within a 95% confidence level. For the verification of this method, a standard reference material with a certified value was analyzed. The analyzed result was in good agreement with the certified value. The isotope dilution liquid chromatography/tandem mass spectrometry result of the human serum was also compared with results obtained from clinical laboratories, and showed inconsistent results. These inconsistent results suggest that standards certified by the proposed reference method are required in order to improve measurement reliability in clinical fields.
A device with direct sampling, dilution, and sample loading has been utilized to couple a vertically hanging immobilized cellulase bioreactor with a high-performance liquid chromatography system to make a successful on-line analysis of the 3 major carbohydrate products: glucose, xylose, and cellobiose in the cellulase hydrolysate. The coupled analysis system for successive on-line monitoring of the enzymatic hydrolysis of paper cellulose was stable over a period of 96 h. A comparative study involving 4 different quantification methods for the 3 carbohydrates was conducted. The internal standard calibration method with on-line dilution was observed to be the best procedure in terms of accuracy, precision, limit of detection, and systematic error reduction. With this method, the analysis for glucose had 99.9% accuracy, RSD below 2.4%, and a 2.1 ppm detection limit. Matrix matching was found important in the preparation of standard calibration curves with refractive index detection.
2-(2,3-Anthracenedicarboximido)cyclohexane derivatives (AC) have been known as the evolutionary diastereomerizing reagents for enantiomer discrimination in HPLC with ODS. However, a substantial separation of diastereomers can be observed only at lower temperatures, such as -40°C. Therefore, in this work, poly(octadecyl acrylate)-grafted silica, ODAn was applied as an alternative stationary phase to ODS for the separation of AC-derived diastereomers. As a result, complete separation was achieved even under the conventional condition: for example, methanol as the mobile phase and 0°C as the column temperature. An investigation on the temperature dependency of the selectivity demonstrated that ODAn shows a remarkable increase in selectivity at temperatures below 30°C, which almost agreed with the peak-top temperature of the endothermic peak in a DSC thermogram for ODA35 immersed in a mobile phase. The better separation would be derived from a highly ordered structure of ODAn and a carbonyl-π interaction with AC-derived diastereomers.
The catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide has been investigated for the fluorescent derivatization of kynurenic acid under conditions with no exposure to light. Non-fluorescent kynurenic acid was converted into a fluorescent compound (Ex: 367 nm, Em: 470 nm) with HRP in the presence of hydrogen peroxide, and the optimum conditions of this fluorescent derivatization were investigated. Moreover, this fluorescent derivatization was developed for a spectrofluorometric determination of trace amounts of kynurenic acid by measuring the fluorescence intensity of the fluorescent compound. The calibration curve obtained was linear from 1.0 to 10.0 nmol of kynurenic acid in a 1.0 mL sample solution. The relative standard deviation at 5.0 nmol of kynurenic acid was 5.71% (n = 5). By adjusting the bandwidths for both the excitation and emission to 15 nm, the calibration curve was also linear in the range between 0.1 to 1.0 nmol of kynurenic acid in a 1.0 mL sample solution. This method was applied to the fluorometric determination of trace amounts of kynurenic acid in the control sera.
A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of 19-nortestosterone (17β-NT) residues in aquaculture tissues. The limit of detection (LOD) was determined to be 0.08 ng g-1 and the limit of quantification (LOQ) was less than 0.8 ng g-1. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS). This proposed technique could be applied to routine residue analysis.
Quantification of 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) was studied by its chemiluminescence (CL) reaction with luminol in an alkaline medium. The stability of DBDMH, 1,3-dichloro-5,5-dimethylhydantoin (DCDMH) and 1-bromo-3-chloro-5,5-dimethylhydantoin (BCDMH) in water was initially assessed by its CL reaction capability. The results indicated that the hydrolysis process was critically dependent on the types of reagents and their pHs. Capillary electrophoresis (CE) separation with CL detection procedure was applied to the DBDMH solution. It was found that at least 3 species in the aqueous DBDMH solution could oxidize luminol to give luminescence: one of them was confirmed to be hypobromite and the others could be the unhydrolyzed or active oxygen produced in the hydrolysis reaction. Finally, a flow-injection chemiluminescent method was proposed for the determination of DBDMH. The concentration of the analyte showed a linear relationship with the CL intensity in the range of 1.2 × 10-10 to 1.0 × 10-6 mol dm-3 and the detection limit was as low as 6.2 × 10-11 mol dm-3. The relative standard deviation (RSD) is 1.7% (n = 9) for 2.8 × 10-7 mol dm-3 DBDMH.
Nanoparticles of cadmium telluride coated with mercaptoacetic acid were prepared in the water phase. Further, an assay of lysozyme with a sensitivity at the nanogram level is proposed. At pH 7.28, lysozyme with positive charges can interact with CdTe nanoparticles. The resonance light-scattering (RLS) signals of functionalized nano-CdTe were greatly enhanced by lysozyme in the region of 300 - 600 nm, characterized with peaks located at 367, 470 and 533 nm. A linear relationship could be established between the enhanced RLS intensity and the lysozyme concentration in the range of 0.06 - 4.0 µg mL-1. The limit of detection was 9.5 ng mL-1. The contents of lysozyme were determined with recoveries of 95.6 - 104.8% and RSD of 1.5 - 2.3%, respectively. This method is sensitive, rapid, accurate and simple, and provides a new and reliable means for the quantity determination of lysozyme.
Multivariate calibration methods (partial least squares calibration, back propagation multilayer perceptrons networks, radial basis functions and generalized regression neural networks) were applied to the simultaneous fluorometric quantification of levofloxacin, garenoxacin and grepafloxacin, without previous separation steps. A data matrix was obtained by registering the emission spectra of mixtures of the three quinolones in urine (with concentrations ranging over 0.00 - 0.40 µg mL-1 for each quinolone) with a 283 nm excitation at pH 4.0. The generalized regression neural network model proved to be the most adequate model for simultaneous quantification of the three quinolones in urine samples.
An automated on-line sample-preparation method using a computer-controlled pretreatment system (Auto-Pret AES system) coupled with ICP-AES was developed. In this work, an iminodiacetate chelating resin, packed in a mini-column and installed in the system was employed for the collection/concentration of 13 trace metals, including such toxic metals as Be, Cd, Cr, Cu and Pb. The limits of detection of the proposed method for trace metals were in the range of 0.001 (Be) - 0.18 (Pb) ng mL-1. The enrichment factors for metal ions were about 19 times, when 5 mL of samples were used. The sample throughput was 11 h-1. The accuracy and the precision of the method were evaluated using river-water reference materials, SLRS-4 from NRCC, JSAC 0301-1 and JSAC 0302 from the Japan Society for Analytical Chemistry. The proposed method can be favorably applied to the collection/concentration of trace metals in natural water samples.
A simple method is described for preconcentration and separation of trace metals such as Ag, Co, Cr, Cu, Fe, Mn, Ni and Pb simultaneously from seawater using a cloud point extraction (CPE) procedure. Triton X-114 nonionic surfactant and ammonium pyrrolidine dithiocarbamate (APDC) have been used as an extraction medium and a chelating extractant, respectively. The amounts of Triton X-114 and APDC and the pH value necessary for extraction were carefully optimized. The preconcentration factor of about 200 is achieved for all the studied metals. Electrothermal atomic absorption spectrometry (ETAAS) with an Ir coated graphite tube as permanent chemical modifier has been used for determination. The limits of detection of Ag, Co, Cr, Cu, Fe, Mn, Ni and Pb were 0.003, 0.008, 0.003, 0.006, 0.015, 0.002, 0.009 and 0.01 ng ml-1, respectively. Certified reference materials such as CASS-4 and NASS-5 (seawater) and NIST-1640 (natural water) have been used for validation of the new method. The relative standard deviation (%) obtained for all the metals are in the range 0.8 - 3.6% for natural water and 11 - 25% for seawater materials, except for Co in NASS-5 for which it was 50%.
Prospects of using different alkaline phosphatases bearing zinc and magnesium ions in their catalytic and allosteric sites, respectively, in pharmaceutical and clinical analysis were demonstrated. Also their application for the determination of zinc in insulin to control injection quality and magnesium in human urine for the diagnosis and treatment of magnesium deficiency was shown. The reaction of p-nitrophenyl phosphate hydrolysis was chosen as an indicator. The choice of appropriate alkaline phosphatase was substantiated, the influence of the nature of buffer solutions on the behavior of the enzyme-metal systems was studied, and the conditions of the indicator reaction proceeding in the presence of sample matrixes were optimized. Simple, rapid, sensitive, and selective enzymatic procedures for determining zinc and magnesium based on their inhibiting and activating effects on the catalytic activity of alkaline phosphatases from seal and chicken intestine, respectively, were developed.
A simple analytical method was established for the simultaneous multielement determination of As, Se and Sb in river water with GF-AAS using TiO2-slurry sampling. Titanium dioxide (TiO2, <0.5 µm) was applied as a sorbent to separate and concentrate these elements in a sample solution (100 - 300 ml); after filtration, a slurry was prepared with the solid phase and 5.0 ml of ultrapure water. In order to apply to river-water samples, the effect of TiO2 in atomization, the effect of organic species of their metal and the interference of foreign ions were investigated. The proposed method was applied to JSAC 0302 and river water.
A 3-mm length of a porous monolithic polymer was prepared in a 0.32-mm inner-diameter fused-silica capillary by an in-situ thermo-polymerization method and used as an on-column frit for a packed capillary HPLC column. The on-column frit can resist high pressure up to 400 bar. A 5-µm packing material was packed in the capillary with the on-column frit by a slurry method. At pressure driving mode, separation of samples was performed using the capillary HPLC column. The in-situ frit preparation method has the advantages of easy preparation, easy control of the location of the frit and a mild preparing reaction condition.
A thin-layer two-phase microcell was fabricated without an adhesive, which showed no fluorescence and resisted acids. The lower hollow of the microcell was thin (0.18 mm), which was in the range of the working distance of the used objective of high magnification (60×) and high numerical aperture (1.2). A reflection method using probe light was newly employed for focusing the objective on a transparent dodecane/water interface strictly with an inverted microscope. The system was applied to in situ fluorescence microscopic measurements of the dynamics of single DNA molecules (165600 base pairs) at the dodecane/water interface.