Flow-through polymerase chain reaction (PCR) microfluidic systems for fast, small-volume DNA amplification on a single chip are significantly impacting medical and bioanalytical research. We have fabricated an improved, practical flow-through PCR chip by weighting a pressure-sensitive polyolefin (PSP) film onto a cyclo-olefin polymer (COP) substrate. The substrate was cut so as to produce microchannels, and was used to amplify DNA using a small moving liquid plug, in contrast to conventional continuous-flow-through PCR. Infrared (IR) imaging-based thermal analysis of the PSP film enabled the gradient and uniformity of the rapidly flowing microfluid to be accurately visualized, because the PSP film is thin, transparent and heat resistant. The liquid plug flow-through PCR, operating at 150 μl min⁻¹, showed approximately 55% amplification compared to a commercial PCR instrument, and required only 250 s for 40 cycles. To our knowledge, this is the fastest cycling time reported to date. In a real test sample, the liquid plug flow-through PCR could detect the presence or absence of anthrax in a sample solution in approximately 250 s. Thus, this liquid plug flow-through PCR system, based on our novel PCR chip, excelled in a practical applications compared to other devices.

View full abstract

A two-dimensionally imaging spectrometer system was employed to obtain the spatial distribution of the excitation temperature in a glow discharge plasma for emission analysis. A Grimm-style glow discharge excitation source, whose hollow anode had an inner diameter of 8 mm, was observed over the whole emission area. The mapping of the excitation temperature was conducted by using a two-line method under the assumption of the Boltzmann distribution. Iron was chosen as the probe element, and a pair of the atomic lines: Fe I 371.99 nm (3.33 eV) and Fe I 375.82 nm (4.25 eV), were employed for the measurement. The excitation temperature was a little varied over the plasma region except for the edge portion, even when the discharge voltage as well as the argon pressure were changed as the experimental parameters. The temperature was estimated to be 4650 – 4950 K at a central portion of the plasma having a 5-mm-diameter circle, implying that the emission excitation at the central portion occurred almost uniformly for each discharge condition.

View full abstract

For point-of-care testing (POCT), a sandwich-typed immunoassay on poly(dimethylsiloxane) (PDMS) microfluidic chips was developed by employing two original technologies: a power-free microchip and laminar flow-assisted dendritic amplification (LFDA). Sequential injection of reagents of immunoassay was carried out by the power-free microchip. In addition, for the amplification of a fluorescent signal, LFDA exploited the interaction between FITC-labeled streptavidin and biotinylated anti-streptavidin on the interface of the two streams of laminar flow during the sequential injection of the reagents of the immunoassay. The microfluidic immunoassay was finished within 21 min with a sample volume of 500 nL. The calibration curve of a quantitative analysis satisfied the concentration range of the cut-off values of prostate-specific antigen (PSA) in serum. The limit of detection (LOD) reached 520 pg/mL (16 pM) of PSA in human female serum. We believe that this microfluidic immunoassay and device are promising for POCT.

View full abstract

In the present work, we found that methylene blue (MB) caused an efficient quenching of the fluorescence of carbon dots (CDots). However, as ct-DNA was added, the fluorescence of CDots was restored reversibly. Under the experimental conditions, CDots were excellent electron donors. MB adsorbed on the surface of CDots and quenched the fluorescence of CDots through an electron-transfer process; and then the addition of DNA caused a restoration of the CDots fluorescence intensity, since DNA could bind with MB and remove it from the CDots. Based on this phenomenon, we developed a new system for DNA detection. A sensitive detection of ct-DNA with a detection limit of 1.0 × 10⁻⁶ mol L⁻¹ and a linear detection range from 3.0 × 10⁻⁶ to 8.0 × 10⁻⁵ mol L⁻¹ with a relative fluorescence intensity of CDots were achieved. To the best of our knowledge, this is the first use of CDots for DNA detection.

View full abstract

The performance of a simple gold (Au)-deposited optical fiber sensor system based on surface plasmon resonance (SPR) is presented. Our sensor system comprises a light-emitting diode (LED) and a photodiode (PD) as the light source and the detector, respectively. The response curves and sensor properties of the Au-deposited optical fiber with a Au film thickness of 45 nm obtained by using three LEDs with 563, 660, and 940 nm emissions were investigated. The sensor properties depend strongly on the wavelength of the incident light and can be controlled by changing the LED emission wavelength. The response curves of the sensor obtained at the three wavelengths and calculated using SPR theoretical equations were compared with those obtained by experimentation. Our sensor system represents a new SPR-based refractometer with performance almost equal to that of an Abbe refractometer. To demonstrate its usefulness, ethanol concentrations in various spirits were measured and the results compared with those as labeled.

View full abstract

Quartz crystal microbalance (QCM) electrodes modified with nano-thin films were used to develop a system for measuring significant environment changes (smoke, humidity, hazardous material release). A layer-by-layer approach was used for the deposition of sensitive coatings with a nanometer thickness on the electrode surface. The QCM electrode was modified with self-assembled alternate layers of tetrakis-(4-sulfophenyl) porphine (TSPP) (or its manganese derivative, MnTSPP) and poly(diallyldimethylammonium chloride) (PDDA). The QCM sensors, which had been reported previously for humidity sensing purposes, revealing a high possibility to recognize significant environmental changes. Identifying of the origin of environmental change is possible via differential signal analysis of the obtained data. The sensors showed different responses to humidity changes, hazardous gas (ammonia) or cigarette smoke exposure. Even qualitative analysis is not yet available; it has been shown that ventilation triggers or alarms for monitoring smoke or hazardous material release can be built using the obtained result.

View full abstract

A capillary chromatography system has been developed based on the tube radial distribution of the carrier solvents using an open capillary tube and a water–acetonitrile–ethyl acetate mixture carrier solution. This tube radial distribution chromatography (TRDC) system works under laminar flow conditions. In this study, a phase diagram for the ternary mixture carrier solvents of water, acetonitrile, and ethyl acetate was constructed. The phase diagram that included a boundary curve between homogeneous and heterogeneous solutions was considered together with the component ratios of the solvents in the homogeneous carrier solutions required for the TRDC system. It was found that the TRDC system performed well with homogeneous solutions having component ratios of the solvents that were positioned near the homogeneous-heterogeneous solution boundary of the phase diagram. For preparing the carrier solutions of water–hydrophilic/hydrophobic organic solvents for the TRDC system, we used for the first time methanol, ethanol, 1,4-dioxane, and 1-propanol, instead of acetonitrile (hydrophilic organic solvent), as well as chloroform and 1-butanol, instead of ethyl acetate (hydrophobic organic solvent). The homogeneous ternary mixture carrier solutions were prepared near the homogeneous-heterogeneous solution boundary. Analyte mixtures of 2,6-naphthalenedisulfonic acid and 1-naphthol were separated with the TRDC system using these homogeneous ternary mixture carrier solutions. The pressure change in the capillary tube under laminar flow conditions might alter the carrier solution from homogeneous in the batch vessel to heterogeneous, thus affecting the tube radial distribution of the solvents in the capillary tube.

View full abstract

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of entecavir in pharmaceutical formulations, using nimesulide as an internal standard. A fused-silica capillary (50 μm i.d.; effective length, 40 cm) was used while being maintained at 25°C; the applied voltage was 25 kV. A background electrolyte solution consisted of a 20 mM sodium tetraborate solution at pH 10. Injections were performed using a pressure mode at 50 mbar for 5 s, with detection at 216 nm. The specificity and stability-indicating capability were proven through forced degradation studies, evaluating also the in vitro cytotoxicity test of the degraded products. The method was linear over the concentration range of 1 – 200 μg mL⁻¹ (r² = 0.9999), and was applied for the analysis of entecavir in tablet dosage forms. The results were correlated to those of validated conventional and fast LC methods, showing non-significant differences (p > 0.05).

View full abstract

An ion chromatography dynamic reaction cell inductively coupled plasma mass spectrometric (IC-DRC-ICP-MS) method for the speciation of arsenic and selenium compounds is described. Chromatographic separation was performed in a gradient elution mode using 0.5 mmol L⁻¹ ammonium citrate in 1% methanol (pH 4.5) and 15 mmol L⁻¹ ammonium citrate in 1% methanol (pH 8.0). The potentially interfering ³⁸Ar⁴⁰Ar⁺ and ⁴⁰Ar⁴⁰Ar⁺ at selenium masses of m/z 78 and 80 were reduced in intensity by approximately 3 orders of magnitude by using 1.0 mL min⁻¹ CH₄ as a reactive cell gas in the DRC. Arsenic was determined as the adduct ion ⁷⁵As¹²CH₂⁺ at m/z 89. The detection limits of the procedure were in the ranges of 0.006 – 0.009 ng As mL⁻¹ and 0.009 – 0.03 ng Se mL⁻¹, respectively. This method has been applied to determine various arsenic and selenium compounds in cereal samples. The accuracy of the method has been verified by comparing the sum of the concentrations of individual species obtained by the present procedure with the total concentration of elements. The arsenic and selenium compounds were quantitatively extracted with a Protease XIV and α-amylase solution in a microwave field at 70°C during a period of 30 min. The spike recoveries were in the range of 94 – 105% for all determinations.

View full abstract

Simple and sensitive spectrofluorometric and HPLC methods for the determination of ribavirin (RIB) were developed. The methods were based on the reaction of the 5′-hydroxyl group of the sugar moiety in RIB with dansyl chloride in a bicarbonate solution (pH 10.5) to form a fluorescent derivative. The first method was based on measuring the fluorescence intensity of the derivative in dichloromethane at 529 nm (excitation at 382 nm). The second method was HPLC separation of the derivative on a reversed-phase C₁₈ column with fluorescence detection. The linear ranges were 200 – 900 and 50 – 1000 ng/mL for the spectrofluorometric and HPLC methods, respectively. The derivatization product was characterized by spectroscopic methods. The proposed methods were successfully applied to analysis of the capsules.

View full abstract

A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the rapid and accurate quantification of zidovudine (AZT) in sustained release tablets during stability testing was developed. A Waters RP-18 XTerra™^® column, using a water:methanol (80:20, v/v%) mobile phase at a flow rate of 1.0 ml min⁻¹, and UV detection at 266 nm, was employed. The method of validation parameters indicate a linear range of between 40.0 to 220.0 μg ml⁻¹ with an LOQ of 1.985 μg ml⁻¹ and an LOD of 0.655 μg ml⁻¹ for the analyte. The degradation products of AZT were isolated and characterized for the first time. There was a very little decline of antiviral by heat, and AZT did not completely degrade either by acid or alkaline hydrolysis. On the other hand, oxidation caused a higher degradation stress in the drug. Finally, the degradation products resulting from stress studies were not found to interfere with the detection of antiviral, which is an advantage of the presently proposed method.

View full abstract

A new method for the determination of the herbicide diuron, using a flow injection manifold and photoinduced chemiluminescence detection, is presented. The pesticide, in basic medium, was irradiated on-line with UV light (254 nm) for 53 s. A short discussion about the possible irradiation products is included. The chemiluminescent response of the photoproducts was induced by oxidation with potassium ferricyanide in phosphate buffer at pH 11.5. The method permitted the quantification of diuron over the 0.1 – 4.0 mg L⁻¹ range, with a detection limit (S/N = 3) of 20 μg L⁻¹ when the method was applied directly. However, the use of solid phase extraction (SPE) performed with C₁₈ cartridges allowed us to achieve a limit of detection of 0.4 μg L⁻¹ and a 1.5 – 30 μg L⁻¹ dynamic range. The method was successfully applied to the diuron determination in samples of water from different sources (spring, ground, mineral, irrigation, sea and tap waters) with a low consumption of reagents.

View full abstract

The chemiluminescence (CL) emission, observed when rhodamine B (RB) in 1-hexanol–cyclohexane was mixed with cerium(IV) sulfate in sulfuric acid dispersed in a reversed micellar medium of cetyltrimethylammonium chloride (CTAC) in 1-hexanol–cyclohexane/water, was investigated using a flow-injection system. The CL emission from the oxidation reaction of RB with Ce(IV) was found to be stronger in the CTAC reversed micellar solution compared with an aqueous solution. Bearing on the enhancement effect of the CTAC reverse micelles on the RB-Ce(IV) CL, several studies including stopped-flow, fluorescence and electron spin resonance (ESR) spectrometries were performed. Rapid spectral changes of an intermediate in the RB-Ce(IV) reaction in the aqueous and reversed micellar solutions were successfully observed using a stopped-flow method. The effect of the experimental variables, i.e., oxidant concentration, sulfuric acid concentration, the mole fraction of 1-hexanol, water-to-surfactant molar concentration ratio, flow rate, upon the CL intensity was evaluated. Under the experimental conditions optimized for a flow-injection determination of RB based on the new reversed micellar-mediated CL reaction with Ce(IV), a detection limit of 0.08 μmol dm⁻³ RB was achieved, and a linear calibration graph was obtained with a dynamic range from 0.5 to 20 μmol dm⁻³. The relative standard deviation (n = 6) obtained at an RB concentration of 3 μmol dm⁻³ was 3%.

View full abstract

Air-segmentation is applied to amplitude-modulated multiplexed flow analysis, which we proposed recently. Sample solutions, the flow rates of which are varied periodically, are merged with reagent and/or diluent solution. The merged stream is segmented by air-bubbles and, downstream, its absorbance is measured after deaeration. The analytes in the samples are quantified from the amplitudes of the respective wave components in the absorbance. The proposed method is applied to the determinations of a food dye, phosphate ions and nitrite ions. The air-segmentation is effective for limiting amplitude damping through the axial dispersion, resulting in an improvement in sensitivity. This effect is more pronounced at shorter control periods and longer flow path lengths.

View full abstract

The present paper describes an inline flow-injection analysis system for the determination of sulfide in water samples, exploiting the Fischer reaction. Water samples were collected and introduced into a reactor of the FIA system. The sulfide released, after sample acidification, was carried out with a nitrogen gas flow and mixed with N,N diethyl-p-phenylenediamine (DEPD) solution in the presence of Fe(III). The blue dye formed was measured in the wavelength range between 672 – 679 nm. An evaluation of the effects of chemical and flow factors was performed using the factorial design of two levels, while optimization was accomplished by a Doehlert matrix. The system presented two linear-response ranges: the first of 0.433 to 400 μg L⁻¹ and the second of 400 to 3500 μg L⁻¹. The detection and quantification limit were found to be 0.130 and 0.433 μg L⁻¹, respectively, while the sample throughput was 12 h⁻¹. The precision was evaluated as the relative standard deviation (n = 10); for 50 and 100 μg L⁻¹ sulfide it was found to be 1.9 and 2.3%, respectively. The method showed satisfactory selectivity regarding the main interference present in environmental samples. The accuracy of the method was successfully evaluated in environmental water samples after a comparison with a literature reference method.

View full abstract

The acetaldehyde-derived cyclic propano adduct of 2′-deoxyguanosine was easily oxidized electrochemically into the cross-linked dimer as an oxidative product. The structural and spectroscopic characteristics of the dimer were investigated by MS, ¹H and ¹³C-NMR, UV, and DFT calculations. The dimer formation was inferred from a molecular ionic peak of m/z 705 ([(2M–2H)+H]⁺, M being the molecular weight of the monomer) on the ESI-MS spectra and the chemical formula as C₂₈H₃₆N₁₀O₁₂ provided by the high-resolution ESI-MS results. The C2–N5 linkage between the two monomers in the dimer was deduced from the ¹H- and ¹³C-NMR spectral results. In addition, the correlations in the 2-dimensional NMR spectra (DQF-COSY and HMBC) were consistently explained by the structure of the C2–N5 cross-linked dimer. UV spectral measurements also support the C2–N5 linking in the dimer formation. The formation of the cross-link dimer as an oxidative lesion of the acetaldehyde-derived cyclic propano adduct of guanosine is expected to interfere with DNA replication and to contribute to acetaldehyde-mediated genotoxicity.

View full abstract

In order to monitor a radioactive nuclide of strontium-90 in seawater around nuclear facilities, a solvent-extraction method for collecting Sr(II) in seawater was examined. A reversed-micellar extraction system containing an anionic surfactant AOT and a molecular extractant N,N,N′,N′-tetra(n-octyl)diglycolamide (TODGA) in n-hexane was chosen for the extraction of Sr(II) from model solutions of seawater containing 0.5 M NaCl (1 M = 1 mol dm⁻³), 0.05 M MgCl₂, and/or 0.01 M CaCl₂. The combination of AOT-forming reversed micelles and TODGA coordinating with Sr(II) as an organic ligand (extractant) was found to be efficient for the extraction of Sr(II) from model solutions. The mechanism of the reversed-micellar extraction system was also considered in the present study.

View full abstract

A single current “emulsion flow” liquid–liquid extraction apparatus has a head with a number of holes from which micrometer-sized droplets of an aqueous phase spout into an organic phase to mix the two liquid phases. For practical use, however, a fatal problem can occur when particulate components in the aqueous phase plug the holes. In the present study, we have succeeded in solving the problem by applying a counter current-type emulsion flow extractor where micrometer-sized droplets of the organic phase are generated.

View full abstract

A method for the on-line absorptiometric determination of trace amounts of boron in steels was developed using an anion-exchange column presorbed with chromotropic acid. The on-line reaction and separation were achieved by controlling the pH in order to accelerate the complex formation in the column by 2.7 and to stabilize the complex at pH 8 for the selective elution of a 1:2 complex and its detection at 350 nm. The effects from the iron matrix in the sample were effectively removed by using EDTA as a masking agent; a low limit for boron detection (3σ) of 0.04 μg g⁻¹ was obtained.

View full abstract

The uric acid (UA) level in (human) serum is an important indicator of several diseases; however, its electrochemical detection is difficult because the oxidation potentials of UA and ascorbic acid (AA) are very close. In this study, we have developed a simple and efficient UA detection method using a copper-modified carbon electrode. The detection principle is based on the selective oxidation of AA by Cu(II), wherein Cu(II) reacts selectively with AA but not with UA. By performing this specific reaction on an electrode surface, we have successfully distinguished the oxidation potential of UA without AA interference.

View full abstract

Fine needle-type glucose sensors with an outer diameter of less that 0.2 mm were fabricated using a low-cost biodegradable γ-polyglutamic acid (PGA) not only as a glucose oxidase (GOx) immobilizing material, but also as a permselective inner membrane. PGA film was prepared on a cellulose acetate-coated Pt–Ir wire electrode, and GOx was covalently immobilized on its surface using water-soluble carbodiimide. The obtained electrode was practically not affected by the existence of electroactive compounds, and provided long-term stability for approximately one month. It also functioned in horse serum with a good linear relationship between the current and glucose concentrations within the range of 2.8 to 22.4 mM.

View full abstract

An on-site colorimetric technique was developed for the routine determination of chromium, iron and copper in bath solutions for the chromium(III) conversion coating. A portable colorimeter with a red-green-blue light emitting diode was used for the absorbance measurements. Iron and copper were determined as Fe(III)-thiocyanate and Cu(I)-bathocuproindisulfonate, respectively. Chromium(III) was determined simultaneously with iron or copper using green and blue light. A correction method of the matrix effect was proposed and its applicability was demonstrated. Analytical errors were within 500, 5 and 0.3 mg L⁻¹ for chromium(III), iron and copper, respectively.

View full abstract