Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.     

Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100 ms using standard APnEOs sample solutions at various concentrations (0–1000 ppb) under optimum conditions. The lower detection limit defined as IC₈₀ is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.     

Sequential injection immunoassay for environmental measurements

Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis.     

A kinetic method for the determination of nitrite by its catalytic effect on the oxidation of chlorpromazine with nitric acid.

A catalytic spectrophotometric method for the determination of trace amounts of nitrite is proposed. In acidic solution, chlorpromazine (CP) is oxidized by nitric acid to form a red compound, which is further oxidized to a colorless compound. The reaction is accelerated by trace amounts of nitrite and can be followed by measuring the absorbance at 525 nm: nitrite ion is regenerated and multiplied by nitric acid. The absorbance of the reaction increased with an increase in the reaction time, reached a maximum and decreased rapidly. Since the time required for the absorbance to reach the maximum decreased with increasing nitrite concentration, this value was used as the measured parameter for the nitrite determination. Under the optimum experimental conditions (2.3 M nitric acid, 1.2 x 10(-5) M CP, 40 degrees C), nitrite can be determined in the range 0-100 microg l(-1). The relative standard deviations (n = 6) are 4.7 and 1.8% for 40 and 100 microg l(-1) nitrite, respectively. The detection limit of this method (3sigma) is 1.2 microg l(-1). This method was successfully applied to a determination of nitrite in natural water samples.     

Simultaneous Determination of Micro Bromide and Iodide by Kinetic Spectrophotometric Method

ABSTRACT

ABSTRACTA new kinetic spectrophotometric method for the simultaneous determination of concentrations of micro bromide and iodide has been proposed. It is based on their catalytic effects on the reaction of m-cresol purple oxidized by potassium periodate in hydrochloride acid medium. The reaction rate was monitored by measuring the decrease in absorbance at 528nm and the increase in absorbance at 455nm. The total difference in absorbance of the sum of bromide and iodide is identical with determination of bromide was carried out after Cr(VI) oxidized I? to I₂, and I₂ was removed by extraction with CCI₄, and the amount of iodide was measured by subtracting the absorbance change of bromide from the total absorbance change in the presence of bromine and iodide. The optimum conditions influencing the reaction rate were studied. The linear range of determination is 0～4.0μg/ml for Br? and 0～3.0 μg/ml for I?. The detection limits are 0.032μg/ml for Br? and 0.059 μ/ml for I?. The method was successfully applied to the determination of micro amounts of bromide and iodide in food and life samples.     

Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB–GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (λ = 412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions.

The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 μM, with detection limits of 0.031 and 0.014 μM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.) < 5.0% (n = 10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.     

Simultaneous spectrophotometric determination of paracetamol and salicylamide in human serum and pharmaceutical formulations by a differential kinetic method

A rapid, simple, and sensitive differential kinetic method is presented for the determinations of acetaminophen (also known as paracetamol) and salicylamide. The method is based on their oxidation reaction by Fe3+ ion in the presence of 1, 10-phenanthroline as indicator. The reactions can be monitored spectrophotometrically by measuring the increase in the absorbance of the solution at 510 nm. Two times were selected one in which only paracetamol is oxidized by Fe3+ ion and the other in which both drugs are oxidized by Fe3+ ion. The data were evaluated by the proportional equations method. The method allowed the simultaneous determination of paracetamol and salicylamide at concentrations between 0.5-20 and 1-40 microg/mL with relative standard deviations of 3.47 and 2.58%, respectively. The method was applied to the simultaneous determination of paracetamol and salicylamide in human serum and pharmaceutical formulations.     

Kinetic determination of thiocyanate on the basis of its catalytic effect on the oxidation of methylene blue with potassium bromate.

This study reports a sensitive kinetic spectrophotometric method for the determination of trace amounts of thiocyanate. In acidic solution, Methylene Blue (MB) is oxidized by bromate to form a colorless compound. The reaction is accelerated by trace amounts of thiocyanate and can be followed by measuring the absorbance at 664 nm. The absorbance of the reaction decreased with an increase in the reaction time. Under the optimum experimental conditions (0.56 M of sulfuric acid, 3.9 x 10(-5) M of MB, 3.0 x 10(-3) M of bromate, 180 s, 25 degrees C), thiocyanate can be determined in the range 5.0 - 180 ng/ml. The relative standard deviations (n = 8) are 2.81 and 1.43% for 10.0 and 150 ng/ml thiocyanate, respectively. The detection limit of this method is (3sigma) 3.8 ng/ml. This method was successfully applied to the determination of thiocyanate in real samples.     

A kinetic method for the determination of copper(II) by its catalytic effect on the oxidation of 3-methyl-2-benzothiazolinone hydrazone with hydrogen peroxide: a mechanistic study.

A catalytic spectrophotometric method for the determination of traces of copper(II) is proposed. 3-Methyl-2-benzothiazolinone hydrazone (MBTH) is oxidized by hydrogen peroxide to form a yellowish-brown compound. The reaction is accelerated by trace amounts of copper(II), and can be followed by measuring the increase in the absorbance at 390 nm. Since the absorbance at 40 min from the reaction start increases with an increase in the copper(II) concentration, the absorbance value is used as a parameter for copper(II) determination. Under the optimum experimental conditions (8.4 x 10(-3) mol dm(-3) MBTH, 0.7 mol dm(-3) hydrogen peroxide, pH 5.2, 35 degrees C), copper(II) can be determined in the range 0-50 microg dm(-3). The relative standard deviations are 6.9, 3.5, 2.7% for 2, 20 and 40 microg dm(-3), respectively. The detection limit of this method (3sigma) is 0.27 microg dm(-3). It was successfully applied to a determination of copper(II) in river water, tap water and ground-water samples. According to the results of a kinetic study, a mechanism is proposed which leads to the following rate equation: R0(cat) = kK1K2[MBTH][H2O2][Cu(II)]0/{(1 + K2[H2O2])[H+]}.     

Development of a spectrophotometric Sequential Injection Analysis (SIA) procedure for determination of ammonium: A Response Surface Methodology (RSM) approach

This paper describes the optimization and use of a Sequential Injection Analysis (SIA) procedure for ammonium determination in waters. Response Surface Methodology (RSM) was used as a tool for optimization of a procedure based on the modified Berthelot reaction. The SIA system was designed to (i) prepare the reaction media by injecting an air-segmented zone containing the reagents in a mixing chamber, (ii) to aspirate the mixture back to the holding coil after homogenization, (iii) drive it to a thermostated reaction coil, where the flow is stopped for a previously established time, and (iv) to pump the mixture toward the detector flow cell for the spectrophotometric measurements. Using a 100 μmol L^− 1 ammonium solution, the following factors were considered for optimization: reaction temperature (25 - 45 °C), reaction time (30 - 90 s), hypochlorite concentration (20 - 40 mmol L^− 1), nitroprusside concentration (10 - 40 mmol L^− 1) and salicylate concentration (0.1 - 0.3 mol L^− 1). The proposed system fed the statistical program with absorbance data for fast construction of response surface plots. After optimization of the method, figures of merit were evaluated, as well as the ammonium concentration in some water samples. No evidence of statistical difference was observed in the results obtained by the proposed method in comparison to those obtained by a reference method based on the phenol reaction.     

Determination of boron as boric acid in eye lotions using a sequential injection system

A simple and reliable sequential injection analysis (SIA) system is described for the determination of boron as boric acid in eye lotions. This method is based on the complexation reaction between d-sorbitol and boric acid followed by the acid-base reaction with methyl orange (MO). The colour change reaction is monitored at 520 nm. The system is able to monitor boron at a frequency of 30 samples per h with a relative standard deviation of less than 0.6%. The calibration graph is linear up to 12-mg l(-1). The system has a detection limit of 0.06-mg l(-1). No significant differences (at 95% probability level) were found between the proposed SIA method and the reference method.     

Selective determination of o-phenylenediamine by surface-enhanced Raman spectroscopy using silver nanoparticles decorated with α-cyclodextrin

Microchimica Acta - We have developed a SERS method for the determination of o-phenylenediamine (OPD) in water. It is based on the chemical cyclization reaction of OPD with nitrite in acidic...     

Highly Sensitive and Selective Determination of Manganese in Tea Leaves by a Catalytic Kinetic Spectrophotometric Method

The catalytic effect of manganese(II) on the oxidation of barium diphenylamine sulfonate (BDAS) with potassium bromate in sulfuric acid was studied. Progress of the reaction was monitored, spectrophotometrically, by measuring absorbance changes at 547.5 nm. A highly sensitive, selective and simple method was accordingly developed for the determination of trace amounts of Mn(II), with no need for a separation or pre‐concentration step. Effects of reaction time, concentration of reagents, temperature and ionic strength on the reaction rate were studied. In the optimized conditions, manganese could be determined in a range of 1 to 60 ng mL^?1 with a detection limit of 0.2 ng mL^?1. The proposed method was successfully applied to the determination of manganese in tea leaves.     

High-performance affinity chromatography of a chick lectin on an adsorbent based on hydrophilic polymer gel

A mouse monoclonal antibody (SIA4-5) which reacts with a chick 14K lectin (C14K) was covalently attached to a new support for high-performance affinity chromatography, TSKgel Tresyl-5PW, which is a preactivated, polymer-based particle. The immobilized antibody (SIA4-5-5PW) thus prepared proved to be useful in measuring not only the molecular properties of C14K but also specific interactions of C14K with SIA4-5 and hapten sugars. The C14K preparation was fractionated according to the oligomeric structure and with slight differences in affinity to SIA4-5 although the former was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Application of the method for quantitative analytical purposes was successful.     

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.     

Spectrophotometric and volumetric molybdenum determination with 2,2'-biquinoxalyl

Volumetric and spectrophotometric methods for molybdenum determination based on reaction with 2,2'-biquinoxalyl (2,2'-BQx) in concentrated hydrochloric acid media have been developed. Absorption spectra of the 1,1'-dihydro 2,2'-biquinoxalylene complex shows the most intensive absorption band at 685 nm with molar absorptivity = 3.3 x 10(4) 1. mole(-1). cm(-1). The compound is characterized by good durability to high temperatures and concentrated acid media. The mentioned indicator gives distinct colour changes at the titration end-point. The spectrophotometric method for molybdenum determination is based on the use of the difference in absorbance between the oxidized and reduced forms of 2,2'-BQx. The indicator is reduced with Sn(II) and then part of it is reoxided as a result of addition of Mo(VI). The difference in absorbance between the blank determination and molybdenum sample increases linearily in the concentration range 0.2-2.0 mug Mo/cm(3). 2,2'-Biquinoxalyl was used as an indicator in the volumetric method for the determination of molybdenum concentrations in steel alloy. The interfering ions FE(III) and Cr(III) are easily eliminated as the precipitate of hydroxides. The mineral acids, hydrochloric sulphuric and perchloric acids, have been tested as reaction media.     

催化褪色光度法测定痕量亚硝酸根

用吸收光谱法研究了在磷酸介质中,亚硝酸根催化甲基绿被溴酸钾氧化而褪色的反应。在紫外可见光谱区,甲基绿的三个吸收峰出现在632．6,421．4和313．4nm。根据亚硝酸根的催化褪色反应在632．6nm产生的吸光度差或在三个吸收峰产生的吸光度差的总和,与亚硝酸根的浓度呈线性关系,建立了催化褪色光度法测定痕量亚硝酸盐的方法。     

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500 ppb) under optimum conditions. The time required for analysis is less than 15 min.     

阻抑动力学光度法测定痕量间二硝基苯

在硫酸介质中,痕量间二硝基苯能灵敏地阻抑Fe3+催化H2O2氧化中性红染料褪色的反应,由此建立了一种测定痕量间二硝基苯的阻抑动力学光度法。采用固定时间法,在λmax=530 nm处,对阻抑体系和非阻抑体系进行光度测定,应用正交试验法确定该阻抑褪色反应的最佳实验条件,并测定了其反应级数。该方法线性范围为0～48μg/L,检出限0.103μg/L,回收率为97.8%～98.9%,测定结果的相对标准偏差为1.5%～2.1%。该方法用于环境水样中间二硝基苯含量的测定,测定结果可靠。