DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection     

Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Highly sensitive spectrofluorimetric determination of trace amounts of lecithin

A new spectrofluorimetric method was developed for the determination of trace amounts of lecithin using the ciprofloxacin (CIP)–terbium (Tb³⁺) ion complex as a fluorescent probe. In a buffer solution at pH=5.60, lecithin can remarkably reduce the fluorescence intensity of the CIP–Tb³⁺ complex at λ=545 nm. The reduced fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. Optimum conditions for the determination of lecithin were also investigated. The linear range and detection limit for the determination of lecithin were 1.0×10⁻⁶–3.0×10⁻⁵ mol L⁻¹ and 3.44×10⁻⁷ mol L⁻¹, respectively. This method is simple, practical, and relatively free of interference from coexisting substances. Furthermore, it has been successfully applied to assess lecithin in serum samples.      

Evaluation of CD detection in an HPLC system for analysis of DHEA and related steroids

The biological importance of dehydroepiandrosterone (DHEA) is reflected by the fact that DHEA is a crucial precursor of the biosynthesis of the steroidal sex hormones. Simultaneous separation of DHEA, dehydroepiandrosterone sulfate (DHEA-S), pregnenolone, androstenedione and testosterone has been accomplished by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) based on isocratic elution applying circular dichroism (CD) detection at 295 nm. Addition of tetrabutylammonium hydrogensulfate to the mobile phase increases the retention of DHEA-S on the C8-silica column by an apparent ion-pairing mechanism without affecting the retention of the other (non-ionic) steroids. CD spectroscopy provides highly selective detection of compounds possessing optically active absorption bands and the separation is even more selective in the higher wavelength range applied. The linearity of the steroid concentration (c, mg mL⁻¹) versus peak area was tested in the concentration range of 0.5–2 mg mL⁻¹ (injected quantities were 10–40 μg). The relative standard deviation (RSD) values for DHEA and DHEA-S indicated a good intra-assay and inter-assay precision of the method.      

A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons

A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250–310 nm, 310–400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associfated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L⁻¹ for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics. Lapainis and Scanlan contributed equally to this work.     

Determination of trace elements in suspensions and filtrates of drinking and surface water by wavelength-dispersive X-ray fluorescence spectrometry

An X-ray fluorescence method (XRF) is presented that allowed low detection limits (at the 0.1–23 ng mL⁻¹ level) to be obtained for Cr, Mn, Fe, Ni, Zn, Sr, Pb, Bi and Br in water. The samples were prepared using a thin layer method. Trace elements were determined via the calibration curve and standard addition. Absorption effects and inhomogenities in prepared samples were checked for using the emission–transmission method and internal standards, respectively. The results from the XRF method were compared with the results from the inductively coupled plasma atomic emission spectrometry method.      

Simultaneous determination of uric acid and ascorbic acid at a ferrocenium–thioglycollate modified electrode

Self-assembled monolayers (SAMS) of chemisorbed thioglycollate on a gold electrode surface have been used as a base interface for the electrostatic adsorption of ferrocenium ion. Electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were used to evaluate the electrochemical properties of the supramolecular film. The bare gold electrode failed to distinguish the oxidation peaks of ascorbic acid (AA) and uric acid (UA) in phosphate buffer solution (PBS, pH 7.0), while the ferricinium–thioglycollate modified electrode could separate them efficiently. In differiential pulse voltammetric measurements, the prepared gold electrode could separate AA and UA signals, allowing the simultaneous determination of AA and UA. Under optimal conditions and within the linear range of 1.0 × 10⁻⁶ to 5.0 × 10⁻⁴ M, the detection limits of AA and UA achieved were 2.0 × 10⁻⁷ and 1.0 × 10⁻⁷ M, respectively. The applicability of the prepared electrode was demonstrated by measuring AA and UA in human urine without any pretreatment. Figure Fabrication process for the modified electrode     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Laser ablation inductively coupled plasma mass spectrometry for direct analysis of the spatial distribution of trace elements in metallurgical-grade silicon

The spatial distribution and concentration of impurities in metallurgical-grade silicon (MG-Si) samples (97–99% w/w Si) were investigated by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The spatial resolution (120 μm) and low limits of detection (mg kg⁻¹) for quality assurance of such materials were studied in detail. The volume-dependent precision and accuracy of non-matrix-matched calibration for quantification of minor elements, using NIST SRM 610 (silicate standard), indicates that LA-ICP-MS is well suited to rapid process control of such materials. Quantitative results from LA-ICP-MS were compared with previously reported literature data obtained by use of ICP-OES and rf-GD-OES. In particular, the distribution of element impurities and their relationship to their different segregation coefficients in silicon is demonstrated. Dedicated to Professor Klaus G. Heumann     

An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube–cross-linked chitosan composite

A simple method has been devised for immobilization of acetylcholinesterase (AChE)—covalent bonding to a multiwall carbon nanotube (MWNT)–cross-linked chitosan composite (CMC)—and a sensitive amperometric sensor for rapid detection of acetylthiocholine (ATCl) has been based on this. Fourier-transform infrared spectroscopy proved that the native structure of the immobilized enzyme was preserved on this chemically clean and homogeneous composite film, because of the excellent biocompatibility and non-toxicity of chitosan. Glutaraldehyde was used as cross-linker to covalently bond the AChE, and efficiently prevented leakage of the enzyme from the film. Because of the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl, with a value of 132 μmol L⁻¹, forming thiocholine, which was then oxidized to produce a detectable and rapid response. Under optimum conditions the amperometric current increased linearly with the increasing concentration of ATCl in the range 2.0–400 μmol L⁻¹, with a detection limit of 0.10 μmol L⁻¹. Fabrication reproducibility of the sensor was good and the stability was acceptable. The sensor is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis. Abstract     

Sensitivity improvement in capillary electrophoresis using organo-aqueous separation buffers and thermal lens detection

It is shown that organo-aqueous separation buffers show much promise when used in capillary electrophoresis separations with photothermal (thermal lens) detection systems. Acetonitrile–water and methanol–water mixtures were selected, as conventionally used in capillary electrophoresis. It is shown that, despite more sophisticated experimental conditions (significant heat outflow from the capillary body) and peak detection, the theoretical ratio of the thermal lens signal for a binary mixture to the thermal lens signal for an aqueous solution (or the corresponding ratio obtained experimentally under bulk batch conditions) can be used to predict the sensitivity of thermal lens detection in capillary electrophoresis. The limits of detection for 2-, 3-, and 4-nitrophenols selected as model compounds in 70% v/v acetonitrile separation buffers are 1×10⁻⁶ M, 1×10⁻⁶ M and 3×10⁻⁷ M, respectively, and are therefore decreased by a factor of six compared to thermal lens detection in aqueous separation buffers. The overall increase in the thermal lens detection sensitivity in a 100% ACN buffer is a factor of 13.      

Investigating the post-chemiluminescence behavior of phenothiazine medications in the luminol–potassium ferricyanide system: molecular imprinting–post-chemiluminescence method for the determination of chlorpromazine hydrochloride

A new post-chemiluminescence (PCL) phenomenon was observed when phenothiazine medications were injected into the reaction mixture after the chemiluminescence (CL) reaction of luminol and potassium ferricyanide had finished. A possible reaction mechanism was proposed based on studies of the kinetic characteristics of the CL, CL spectra, fluorescence spectra, and on other experiments. The feasibility of determining various phenothiazine medications by utilizing these PCL reactions was examined. A molecular imprinting–post-chemiluminescence (MI-PCL) method was established for the determination of chlorpromazine hydrochloride using a chlorpromazine hydrochloride-imprinted polymer (MIP) as the recognition material. The method displayed high selectivity and high sensitivity. The linear range of the method was 1.0×10⁻⁸∼1.0×10⁻⁶, with a linear correlation coefficient of 0.9985. The detection limit was 3×10⁻⁹ g/ml chlorpromazine hydrochloride, and the relative standard deviation for a 1.0×10⁻⁷ g/ml chlorpromazine hydrochloride solution was 4.0% (n=11). The method has been applied to the determination of chlorpromazine hydrochloride in urine and animal drinking water with satisfactory results.      

Surface modification of amine-functionalised graphite for preparation of cobalt hexacyanoferrate (CoHCF)-modified electrode: an amperometric sensor for determination of butylated hydroxyanisole (BHA)

A cobalt hexacyanoferrate (CoHCF)-modified graphite paraffin wax composite electrode was prepared by a new approach. An amine-functionalised graphite powder was used for the fabrication of the electrode. A functionalised graphite paraffin wax composite electrode was prepared and the surface of the electrode was modified with a thin film of CoHCF. Various parameters that influence the electrochemical behaviour of the modified electrode were studied by varying the background electrolytes, scan rates and pH. The modified electrode showed good electrocatalytic activity towards the oxidation of butylated hydroxyanisole (BHA) under optimal conditions and showed a linear response over the range from 7.9 × 10⁻⁷ to 1.9 × 10⁻⁴ M of BHA with a correlation coefficient of 0.9988. The limit of detection was 1.9 × 10⁻⁷ M. Electrocatalytic oxidation of BHA was effective at the modified electrode at a significantly reduced potential and at a broader pH range. The utility of the modified electrode as an amperometric sensor for the determination of BHA in flow systems was evaluated by carrying out hydrodynamic and chronoamperometric experiments. The modified electrode showed very good stability and a longer shelf life. The modified electrode was applied for the determination of BHA in spiked samples of chewing gum and edible sunflower oil. The advantage of this method is the ease of electrode fabrication, good stability, longer shelf life, low cost and its diverse application for BHA determination. Figure Cyclic Voltammogram of () CoHCF modified electrode, () in presence of 1.9 x 10⁻⁵ M of BHA and () bare electrode, () in the presence of 1.9 x 10⁻⁵ M of BHA in 1.0 M NaCl, pH 7.0     

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs     

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

A novel thiocyanate-selective electrode based on a zinc–phthalocyanine complex

A novel thiocyanate (SCN⁻)-selective PVC membrane electrode based on a zinc-phthalocyanine (ZnPc) complex as neutral carrier is described. The membrane electrode containing ZnPc with 5.1% (w/w) ionophore, 29.2% (w/w) PVC, and 65.7% (w/w) 2-nitrophenyl octyl ether (o-NPOE) as plasticizer displayed an anti-Hofmeister selectivity sequence , and exhibited near-Nernstian potential response to thiocyanate ranging from about 1.0×10⁻¹ to 1.0×10⁻⁶ mol L⁻¹ with a detection limit of 7.5×10⁻⁷ mol L⁻¹ and a slope of 58.1±0.5 mV per decade in pH 3.0 phosphate buffer solution at 25 °C. This preferential response is believed to be associated with the unique coordination between the central metal of the carrier and thiocyanate.      

Small-volume fiber-optic evanescent-wave absorption sensor for nitrite determination

A novel small-volume fiber-optic evanescent-wave absorption sensor based on the Griess–Ilosvay reaction has been developed and evaluated for nitrite determination. The sensor was constructed by inserting a decladded optical fiber into a transparent capillary to form an annular column microchannel. The Evanescent wave (EW) field produced on the optical fiber core surface penetrated into the surrounding medium and interacted with the azo dye, which was generated by the reaction of nitrite and nitrite-sensitive reagents. The detector was designed to be parallel to the axis of the optical fiber. The defined absorbance was linear with the concentration of nitrite in the range from 0.05 to 10 mg L⁻¹, and the detection limit was 0.02 mg L⁻¹ (3σ) with the relative standard deviation (RSD) of 2.6% (n = 8). The present sensor was successfully used to determine nitrite in real samples of mineral water, tap water, rain water, and seawater. The results were consistent with the data obtained by standard spectrophotometric method, showing potential of the proposed sensor for practical application.      

Adsorptive stripping voltammetric determination of netilmicin in the presence of formaldehyde

A linear sweep adsorptive stripping voltammetric method for the determination of netilmicin in the presence of formaldehyde has been proposed for the first time. In the presence of 3.0×10⁻³ g ml⁻¹ formaldehyde, netilmicin exhibits a sensitive cathodic peak at −1.30 V (vs. the saturated calomel electrode, SCE) in a medium of Britton–Robinson buffer (pH 8.7) with a scan rate of 100 mV s⁻¹ after a preconcentration period of 120 s at −1.10 V (vs. SCE). The peak current showed a linear dependence on the netilmicin concentration over the range 4.2×10⁻⁹–1.0×10⁻⁷ g ml⁻¹. The achieved limits of detection and quantitation were 1.0×10⁻¹⁰ and 3.3×10⁻¹⁰ g ml⁻¹ netilmicin, respectively. It was deduced from the experiments that the amine–aldehyde condensation product formed between netilmicin and formaldehyde is mainly responsible for the appearance of the peak. The electrochemical behavior of netilmicin in the presence of formaldehyde has been studied. The method was applied to the direct determination of netilmicin in injectable formulations and spiked human urine and serum samples.      

The electrochemical behavior of p-benzenediol on a self-assembled monolayers Pt electrode modified with N-(2-mercapto-1,3,4-thiadiazol-5-yl)-N′-(4-substituted-arylacetyl) urea

Two novel N-(2-mercapto-1,3,4-thiadiazol-5-yl)-N′-(4-substituted-arylacetyl) urea compounds have been synthesized, characterized by NMR and MS, and used as self-assembly reagents to form self-assembled monolayers (SAMs) on Pt electrodes. The modified electrodes were characterized by electrochemical methods. The electrochemical behavior of p-benzenediol at the SAMs electrodes was investigated. It was found that the electrochemical response to p-benzenediol is controlled by diffusion and can be electrocatalyzed to obtain more symmetrical redox peaks and higher voltammetric current response at the SAMs electrodes, with a peak separation of 80 mV. For p-benzenediol the process at the SAMs electrodes is quasi-reversible with a rate constant of 0.6742 s⁻¹. The SAMs electrodes have been used to determine p-benzenediol by differential pulse voltammetry. The peak current was linear for concentrations of p-benzenediol in the range 1×10⁻⁷−5×10⁻⁴ mol L⁻¹ and the detection limit was 4.0×10⁻⁸ mol L⁻¹. The SAMs electrodes were used to determine p-benzenediol in real photographic developer and in a synthetic waste water sample; the standard addition recovery was in the range 96.6–100.4%.      

Comparison of two solid-phase microextraction methods for the quantitative analysis of VOCs in indoor air

Competitive adsorption on adsorptive solid-phase microextraction (SPME) fibres implies careful determination of operating conditions for reliable quantitative analysis of VOCs in indoor air. With this objective, two analytical approaches, involving non-equilibrium and equilibrium extraction, were compared. The average detection limit obtained for GC-MS analysis of nine VOCs by the equilibrium method is 0.2 μg m⁻³, compared with 1.9 μg m⁻³ with the non-equilibrium method. The effect of the relative humidity of the air on the calibration plots was studied, and shown to affect acetone adsorption only. Hence, the concentrations that can be accurately determined are up to 9 μmol m⁻³. The methods were then applied to indoor air containing different concentrations of VOCs. The non-equilibrium method, involving short extraction time, can be used for detection of pollution peaks whereas equilibrium extraction is preferable for measurement of sub-μg m⁻³ ground concentration levels.