A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a bio-composite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an o-aminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.
An HPLC method with fluorescence detection for the determination of nitric oxide (NO) in cultivated plant cells (Agave pacifica, Agavaceae) was developed. NO was derivatized in situ with 2,3-diaminonaphthalene (DAN) as a labeling reagent and converted to 1(H)-naphthotriazole. The maximum peak height of the derivative was observed by incubation for 3 h at 25°C with 0.2 mM DAN. Excess reagent in cells was removed by washing 3 times with 5 ml of water. The calibration curve for authentic standard of DAN-NO spiked to cultivated plant cells showed a good linearity (r = 0.995) in the range of 5.0 to 50 pmol/g cell. The detection limit at a signal-to-noise ratio of 3 was 3.4 pmol/g cells. The proposed method was successfully applied to the monitoring of NO concentration with cell growth. The effect of thermal treatment on the concentration of NO in plant cells was also examined. The concentration of NO in cells treated at 5°C for 1 h was significantly higher than that treated at 25°C and 35°C for 1 h (n = 3, p < 0.05).
The metallothioneins (MT) self-assembled monolayer modified gold electrode (MT/Au) is demonstrated to catalyze the electrochemical response of dopamine (DA) by cyclic voltammetry. A pair of well-defined redox waves was obtained and the calculated standard rate constant (ks) is 6.97 × 10-3 cm s-1 (20°C) at the self-assembled electrode. The electrode reaction is a quasi-reversible process. The oxidation peak of DA can be used to determine the concentration of DA. The peak current and the concentration of DA follow a linear relationship in the range of 2.0 × 10-5 M to 8.0 × 10-4 M. The detection limit is 6.0 × 10-6 M. By ac impedance spectroscopy, the apparent electron transfer rate constant (kapp) of Fe(CN)63-/Fe(CN)64- at the MT/Au electrode was obtained as 2.0 × 10-5 cm s-1. The MT/Au was characterized with grazing angle FT-IR spectroscopy and contact angle goniometry.
The electrochemical impedance of an iron electrode often shows the capacitive and inductive loops on the complex plane. The capacitive loop originates from the time constant of the charge transfer resistance and the electric double layer capacitance. The inductive loop is explained by Faradaic processes involving the reaction intermediate. In some cases, these loops deviate from a true semicircle. In this paper, the origins and curve-fitting methods for the deviated loops of electrochemical impedance are discussed. The constant phase element (CPE) was used to present the deviation of the capacitive loop instead of electric double layer capacitance. The reaction rate constants, which are a function of the frequency, are proposed for the Faradaic impedance to present the deviated inductive loop.
The electrochemical reduction mechanism of ethidium bromide was first studied by spectroelectrochemistry. This reduction was proved to be a two-step process by cyclic voltammetry, differential pulse voltammetry and spectroelectrochemistry, in which each step was proved to be a one-electron transfer process by a spectropotentiostatic fluorescence technique. Hydroethidine was confirmed to be the final product by comparing the spectrum of the product of the electrochemical reduction to that of the product of the chemical reduction of ethidium bromide, and a carbon-centered radical was concluded to be a reasonable intermediate product during the electrochemical reduction of ethidium bromide.
A speciation scheme of trace molybdenum was proposed for river water based on size fractionation by filtration and ultrafiltration and the catalytic spectrophotometric determination of the reactive molybdenum concentration (CR). The total concentration (CT) of molybdenum was determined by the same method after acid decomposition to obtain the concentration (CT - CR) of unreactive molybdenum. Most molybdenum in natural river-water samples was found to be reactive species. A large part of the molybdenum was found in the fraction of molecular weight (MW) < 103, and was estimated to be MoO42- from the chemical equilibria of molybdate ions. The residual part of molybdenum was found in the colloidal and particle fractions (MW ≥ 104), and was characterized as reactive molybdenum adsorbed or complexed on humic iron aggregates. The coexistence of silicate contributed to a decrease of the particle size of humic iron aggregates associated with molybdenum. The above-mentioned speciation results were confirmed by an analysis of artificial samples. The changes in the fractionation results by acidification (0.1 M HCl) were also used to characterize molybdenum in natural water.
An analytical method using pentafluorobenzyl bromide (PFBB) derivatization and gas chromatography/mass spectrometry (GC/MS) has been applied to identify and quantify chloro-, bromo- and dichlorophenols in air, water and sediment samples. Phenols in air sample were collected with a PS-2 Sep-PAK™ cartridge, and eluted with 2-propanol. For water and sediment samples, liquid-liquid extraction with dichloromethane was carried out, and the solvent was exchanged to 2-propanol. The phenols in the solution reacted with PFBB to form the corresponding pentafluorobenzyl esters. After extracting the derivatives into hexane, the determination was carried out by GC/MS with selected-ion monitoring. The detection limits of phenols in air, water and sediment were 0.0033 - 0.0073 µg/m3, 0.0066 - 0.0147 µg/L and 0.33 - 0.73 µg/kg, respectively. More than 90% recoveries of the halogenated phenols were obtained from real environmental samples spiked by the halogenated phenols. The three isomers of mono-chlorophenols were detected in sediment samples in the range of 5.2 - 9.2 µg/kg in wet weight basis.
A high-performance liquid chromatographic method was developed for the analysis of capsaicinoid compounds, the pungent principles of capsicum fruits. A sequential simplex method was applied to optimize the chromatographic response function used to assess the quality of separation by varying the chromatographic parameters. The separation was achieved in 11 min using a C-8 column of 15-cm length and 4.6 mm diameter using a UV detector. A flow rate of 1.15 ml min-1 at a column temperature of 43.5°C using 63.7% methanol in water gave the most efficient separation. The method was found to be suitable for the determination of the major capsaicinoid compounds in the capsicum samples.
A simple, precise, sensitive and accurate method was developed for rapid determination of trace quantities of iodate. The method is based on the accelerating effect of iodate on the reaction of bromate and chloride acid in the presence of hydrazine in acidic media. The decolorization of Methyl Orange with the reaction products was used to monitor the reaction spectrophotometrically at 525 nm. Iodate could be determined in the concentration ranges of 0.03 - 1.2 µg ml-1. The relative standard deviation for ten replicate determinations of 0.3 µg ml-1 of iodate was 1.65%. The proposed method was applied to the determination of iodate in table salts with satisfactory results.
Two simple and sensitive indirect spectrophotometric methods for the assay of propranolol hydrochloride (PPH) and piroxicam (PX) in pure and pharmaceutical formulations have been proposed. The methods are based on the oxidation of PPH by a known excess of standard N-bromosuccinimide (NBS) and PX by ceric ammonium sulfate (CAS) in an acidic medium followed by the reaction of excess oxidant with promethazine hydrochloride (PMH) and methdilazine hydrochloride (MDH) to yield red-colored products. The absorbance values decreased linearly with increasing concentration of the drugs. The systems obeyed Beer’s law over the concentration ranges of 0.5 - 12.5 and 0.3 - 16.0 µg/ml for PPH, and 0.4 - 7.5 and 0.2 - 10 µg/ml for PX with PMH and MDH, respectively. Molar absorptivity values, as calculated from Beer’s law data, were found to be 1.36 × 104 and 2.55 × 104 l mol-1 cm-1 for PPH, and 2.08 × 104 and 2.05 × 104 l mol-1 cm-1 for PX with PMH and MDH, respectively. The common excipients and additives did not interfere with their determinations. The proposed methods have been successfully applied to the determinations of PPH and PX in various dosage forms. The results obtained by the proposed methods compare favorably with those of official methods.
A novel strategy for exploiting ion exchange in sequential injection systems is proposed. The procedure is based on the selection of a defined volume of a resin suspension, which is introduced and packed in the analytical path, establishing a resin mini-column in the system. The passage of a selected sample volume through the resin mini-column leads to the retention of the analyte, while the sample matrix is discarded. The analyte is eluted during the passage of the eluant/reagent by the packed beads, being the analytical signal monitored (absorbance) in the liquid phase. The beads are then aspirated back to the holding coil and directed to a recovery flask, linked at the selection valve; then the system is ready to begin a new cycle. With the proposed strategy, the main characteristics of the sequential injection system are kept as any new artifact is added to the manifold and system reconfiguration is not required. The feasibility of the approach is demonstrated by the phytic acid determination in food samples. For this specific application, AG1-X8 was selected as ion exchanger, and a solution containing Cl- and Fe(III)-salicylate complex was used as eluant and spectrophotometric reagent.
Measurements of DSC, NMR and high-frequency spectroscopy have been done on PEO-H2O and PEO-H2O-HQ mixed systems. DSC measurements show that water molecules contained in PEO are divided roughly into two species: freezable water and non-freezable water. By 1H-NMR measurements, PEO and H2O protons show an absorption peak around 3.6 ppm and 4.5 ppm, respectively. All the PEO-H2O samples containing H2O show NMR absorption signals, suggesting the existence of movable H2O. Samples with H2O molar ratio over 0.5, show one absorption peak of H2O protons, and more than two peaks of PEO. The results from DSC and NMR measurements show that the bound state of H2O in PEO, as well as the mobility of PEO itself, varies according to the water content in PEO-H2O samples. On the other hand, DSC measurements give the result that PEO-H2O-HQ systems can be considered as homogeneous so long as the content of H2O and/or HQ is not so high. Furthermore, the water molecules in the system exist as bound water. Results of high-frequency spectroscopic measurements for the PEO-H2O and PEO-H2O-HQ systems show good agreement with those of DSC and NMR measurements.