Use of soybean peroxidase in chemiluminescent enzyme-linked immunosorbent assay

A procedure for the production of conjugates of soybean peroxidase (SbP) oxidized by sodium periodate and anti-mouse IgG antibody (Ab) was optimized. A sandwich chemiluminescent enzyme-linked immunosorbent assay (ELISA) for determination of mouse IgG using SbP and specific Ab was developed, and SbP-catalyzed oxidation of luminol was carried out in the absence of any enhancer. Comparison of conjugates produced by labeling Ab by soybean and horseradish peroxidases in the chemiluminescent ELISA showed that in the case of SbP a rate of emission decay formed through luminol oxidation was significantly lower. Application of the soya enzyme allowed the development of the immunoassay with improved sensitivity and a wider linear range.     

Chemiluminescence investigation of detection of rutin in medicine and human urine using controlled-reagent-release technology.

A novel continuous-flow sensor based on chemiluminescence (CL) detection was developed for the determination of rutin in pharmaceutical preparations and human urine by controlled-reagent-release technology. The analytical reagents involved in the CL reaction, including luminol and hexacyanoferrate(III), were both immobilized on an anion-exchange column in a flow-injection system. The CL signal produced by the reaction between luminol and hexacyanoferrate(III), which were eluted from the column through sodium phosphate injection, was decreased in the presence of rutin. CL intensity was inhibited by rutin; the decrement of CL intensity was linear over the logarithm of the rutin concentration range of 1.0-400 ng x mL(-1), and the detection limit was 0.35 ng x mL(-1) (3 sigma). The whole process, including sampling and washing, could be completed in 1.5 min with a relative standard deviation of <3.5%. The flow sensor showed remarkable stability and could be easily reused >450 time; the sensor proposed was applied successfully to the determination of rutin in pharmaceutical preparations and human urine.     

Comparison of enzyme-linked immunosorbent assays with chemiluminescent and colorimetric detection for the determination of ochratoxin A in food

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the determination of ochratoxin A (OTA) was developed using soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) as a detection system. By varying the concentrations of the capture monoclonal anti-OTA antibody, a conjugate of OTA with SbP, and the composition of blocking buffers, the conditions of the immunoassay were optimized. Advantages of CL-ELISA were demonstrated by comparison with ELISA with colorimetric detection (COL-ELISA). The values of IC??, IC??, and working range (IC??-IC??) for CL-ELISA and COL-ELISA were 0.01, 0.08, and 0.02-0.3 ng/mL and 0.08, 0.58, and 0.17-2.2 ng/mL, respectively. The recovery values of CL-ELISA from three soybean spiked samples with OTA concentrations of 0.07, 0.1, and 0.15 ng/mL ranged from 72 to 125%. Determination of OTA in 21 various agricultural commodities showed that OTA in 8 examined samples was not detected by COL-ELISA. Furthermore, it was found that in 4 of these 8 samples the developed CL-ELISA determined OTA at levels from 0.96 to 4.64 ng/g.     

A new green analytical procedure for monitoring sub-nanogram amounts of chlorpyrifos on fruits using flow injection chemiluminescence with immobilized reagents

A novel green method using flow injection chemiluminescence with controlled-reagent-release technology has been investigated for the rapid and sensitive monitoring of sub-nanogram amounts of chlorpyrifos. The analytical reagents involved in chemiluminescence (CL) reaction, luminol and periodate, were both immobilized on an anion-exchange column. The CL signals produced by the reaction between luminol and periodate, which were eluted from the column through water injection, were decreased in the presence of chlorpyrifos. The decrease of CL intensity was linear over the logarithm of concentration of chlorpyrifos ranging from 0.48 to 484.0 ng x mL(-1) (r(2) = 0.9969), and the limit of detection was 0.18 ng x mL(-1) (3sigma). At a flow rate of 2.0 mL x min(-1), the determination of chlorpyrifos, including sampling and washing, could be performed in 0.5 min with a relative standard deviation of less than <3.0%. The proposed method was applied successfully in an assay of remnant chlorpyrifos on fruits such as orange and shaddock with the recovery of 94.4-107.4%. The change of the concentration of chlorpyrifos in a water sample was also investigated, and the variation rate was 99.96% during 35 h in the open air.     

玉米赤霉烯酮化学发光免疫分析检测系统设计

为了保障粮食安全,该研究根据玉米赤霉烯酮抗原抗体反应,以及辣根过氧化物酶催化鲁米诺过氧化氢反应产生化学发光,设计一款应用于粮食行业的玉米赤霉烯酮检测系统。采用侧窗型高精度光电倍增管MD983以及16位AD转换芯片,实现化学发光强度信号的准确测量;步进电机驱动旋转精密转台,通过优化步进电机的S型脉冲驱动控制曲线参数,完成转台的高精度定位控制,实现光电倍增管的测试窗口和化学发光孔精确对准;通过精密直线导轨滑台驱动加样器的进给,实现反应液微米量级的准确微量加样。利用竞争性免疫分析方法,使得赤霉烯酮毒素为0 μg/kg情况下,化学发光反应具有最大发光量,解决真菌毒素低浓度情况下的检测精度难题。经过试验验证,系统检出限为0.1 μg/kg,样品加标回收率在90%以上,标准曲线决定系数为0.996 5,系统检测玉米赤霉烯酮的线性范围为0~60 μg/kg。研究表明建立的玉米赤霉烯酮检测系统满足国家粮食行业对于粮食中玉米赤霉烯酮含量检测要求,为真菌毒素检测仪器的国产化提供参考。     

Determination of malachite green residues in fish using a highly sensitive electrochemiluminescence method combined with molecularly imprinted solid phase extraction

An electrochemiluminescence (ECL) inhibition method combined with molecularly imprinted solid phase extraction (MISPE) was developed for quantitative determination of malachite green (MG) residues in fish. It was found that MG could strongly inhibit the ECL signal of luminol. Under the optimized conditions, the quenched ECL intensity versus the logarithm of the concentration of MG was in good linear relationship over a concentration range from 20 to 5000 ppt. The method detection limit was found to be about 6 ppt. Molecularly imprinted polymers (MIPs) were synthesized as solid phase extraction (SPE) sorbents, and MISPE was used for the selective extraction and purification of MG. By carrying out the oxidation reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which could convert leucomalachite green (LMG) into MG, this method was successfully applied to determine MG residues in fish. A possible mechanism for the quenching effects of MG on luminol was also proposed.     

Development of time-resolved chemiluminescence for the determination of antu in river water, wheat, barley, and oat grain samples

A novel chemiluminescence method for the determination of antu has been developed based on the reaction between potassium permanganate in acid medium with this rat-poison in the presence of formaldehyde as an emission enhancer. The main feature of the system used is that the recording of the whole chemiluminescence intensity-vs-time profiles can be obtained, using the stopped-flow technique in a continuous-flow system. This enables the use of three quantitative parameters adjustable via software settings, one of them a typically kinetic parameter, such as rate of the light-decay reaction, and the others conventional parameters, such as maximum emission intensity and total emission area, which are proportional to the analyte concentration. The optimum chemical conditions for the chemiluminescence emission were investigated. The effect of common emission enhancers, such as formic acid, formaldehyde, glutaraldehyde, acetaldehyde, quinine, fluorescein, rhodamine B, and rhodamine 6G, was studied. The parameters selected were sulfuric acid 4.0 mol L(-)(1), permanganate 0.1 mmol L(-)(1), and formaldehyde 1.0 mol L(-)(1). The calibration graphs obtained with each one of the measurement parameters were linear for the concentration range from 0.05 to 3.00 microg mL(-)(1). The detection limits ranged from 0.005 to 0.010 microg mL(-)(1), and RSD values (n = 10) of 0.99-1.79% at a 0.30 microg mL(-)(1) concentration level and 1.71-2.22% at a 1.0 microg mL(-)(1) concentration level were obtained. The present chemiluminescence procedures were applied to the determination of antu in different kinds of samples, such as river water, wheat, barley, and oat grain samples. Recovery values not significantly different from the spiked amount were found for these determinations.     

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).     

Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of lipid hydroperoxides of linoleic acid: a comparative study against BHT and melatonin.

The antioxidant activity of capsaicin, as compared to BHT and melatonin, was determined by the direct measurement of lipid hydroperoxides formed upon linoleic acid autoxidation initiated by AIBN. The formation of four isomeric lipid hydroperoxides was detected after reverse-phase HPLC separation. Data from three detectors, UV absorption, glassy carbon electrode electrochemical detection, and postcolumn chemiluminescence using luminol, were compared. Capsaicin was more effective than melatonin in suppressing the formation of lipid hydroperoxides but not as effective as BHT. The formation of capsaicin and BHT dimers was observed during oxidation, and the dimers were characterized using APCI MS(n).     

A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria

A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.     

Characterization of betacyanin oxidation catalyzed by a peroxidase from Beta vulgaris L. roots

A protein fraction with peroxidase (EC 1.11.1.7) activity against guaiacol from Beta vulgaris L. roots oxidized both betanidin and betanin (betanidin 5-O-beta-D-glucoside), the former being the more efficient substrate for the enzyme. The protein fraction contained three strongly basic perxidase isoenzymes. Betanidin quinone was formed as the only product in the course of enzymatic betanidin oxidation, whereas betalamic acid and several oxidized cyclo-DOPA 5-O-beta-D-glucoside polymers were generated during the oxidation of betanin. In accordance with the catalytic properties of peroxidase, a possible mechanism for betanidin oxidation is proposed. This mechanism includes the formation of a betanidin radical, which, by further dismutation, yields betanidin quinone and betanidin. The betanidin oxidation rate showed a Michaelis-type dependence on the substrate concentration. The apparent K(M) for the reaction was 0.46 mM. On the basis of the spectral properties of the enzyme responsible for both betanidin and betanin oxidations, its peroxidase nature is suggested.     

Catalytic characteristics of peroxidase from wheat grass

The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).     

Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radical-scavenging ability of aqueous extracts of Food constituents

With the availability of an ultraweak chemiluminescence analyzer, it is possible to monitor the production of a specific oxygen-derived reactive species, such as hydroxyl radical ((*)OH), whenever a suitable chemiluminescent probe is obtainable. Reported herein is the development of a rapid and specific method for detecting (*)OH production using a specific probe, indoxyl-beta-glucuronide (IBG), a low-level chemiluminescence emitter. Using the Fenton reagent as a source of (*)OH, it was shown that IBG could elicit a very strong intensity of chemiluminescence (CL) (16200 +/- 200 photon counts/s). Conversely, IBG was shown to be insensitive to either superoxide radical or hydrogen peroxide with their CL intensities nearly close to the background values (25 +/- 5 and 180 +/- 20 photon counts/s, respectively). Furthermore, it was also shown that this IBG-based CL production could be effectively quenched by the addition of (*)OH scavengers such as sodium salicylate, dimethyl sulfoxide, and penicillamine to the assay system. Taken together, these data indicate that IBG is a specific CL probe suitable for monitoring the production of (*)OH. This system demonstrated inhibitory activities of various aqueous extracts of food constituents on the CL of hydroxyl radicals generated by Fenton's reagents with the order of scavenging efficiencies being Prunus mume > Cordyceps sinensin > Lilium lancifolium > Astragalus membranceus.     

Oxidation of resveratrol catalyzed by soybean lipoxygenase

In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.     

Recent and future advances in air pollution instrumentation

Currently used instruments for the measurement of O₃, SO₂, and the oxides of nitrogen are briefly described. The first utilizes the chemiluminescence of the gas phase reaction of O₃ with ethylene, the second uses the chemiluminescence of excited diatomic sulfur formed in the cool hydrogen flame by reaction between SO₂ and H atoms, while the third depends upon the chemiluminescent reaction NO+O₃. Nitrogen dioxide is thermally dissociated into NO and measured as such. Newer instruments, now under development, that are described include (1) a photofragment detector for NO₂, operating on photolytic cleavage to yield O atoms, which are then measured via the luminescence of their reaction with an excess of added NO, and (2) the UV fluorescence instrument of Okabe.     

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

Evaluation of antiradical capacity by H2O2-hemin-induced luminol chemiluminescence

This paper describes a screening method for antioxidant potential determination based on luminol/hemin/hydrogen peroxide chemiluminescence. The emission depletion, caused by an antiradical compound added during the chemiluminescence decay, is proportional to the number of reactive species trapped. Therefore, the difference between the areas of the emission decay curves, obtained in the absence and in the presence of the potential antioxidant, is a measure for the antiradical capacity of the sample. The technique has been applied to measure the antiradical capacity of pure compounds and complex mixtures from natural origin, providing reliable results that indicate the method's feasibility.     

Development of an analytical method for the determination of beta2-agonist residues in animal tissues by high-performance liquid chromatography with on-line electrogenerated [Cu(HIO6)2]5- -luminol chemiluminescence detection

A novel method was developed for the simultaneous determination of beta2-agonist residues such as terbutaline, salbutamol, and clenbuterol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the enhancement effect of beta2-agonists on the CL reaction between luminol and the complex of trivalent copper and periodate ([Cu(HIO6)2]5-), which was on-line electrogenerated by constant current electrolysis. The HPLC separation used a Nucleosil RP-C18 column (250 mm x 4.6 mm i.d., 5 microm; pore size, 100 A) with a mobile phase consisting of 90% acetonitrile and 10% aqueous ammonium acetate (20 mmol L-1, pH 4.0) at a flow rate of 1.0 mL min-1. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Liver samples were hydrolyzed with beta-glucuronidase followed by a solid-phase extraction procedure using Waters OasisMCX cartridges. Under optimum conditions, the limits of detection at a signal-to-noise ratio of 3 ranged from 0.007 to 0.01 ng g-1 and the limits of quantification at a signal-to-noise ratio of 10 ranged from 0.023 to 0.033 ng g-1 for three beta2-agonists. The relative standard deviations (RSDs) of intra- and interday precision were below 4.5%. The average recoveries for beta2-agonists (spiked at the levels of 0.05-5.0 ng g-1) in pig liver ranged from 84 to 110%, and the RSDs of the quantitative results were from 1.6 to 7.2%. The proposed method was successfully applied to the determination of beta2-agonist residues in pig liver samples.     

小麦种子过氧化物酶的纯化及其部分酶学性质研究

小麦全面粉水抽提液经醋酸酸沉淀,上清再经硫酸铵35%-75%分组沉淀,沉淀溶于pH5.5的乙酸-乙酸钠缓冲液,经FPLC-SOURCE 30S 阳离子交换层析,FPLC-Superdex G-75凝胶过滤层析,纯化了小麦种子过氧化物酶（Wheat Seed Peroxidase）,纯化酶的比活力为3912U／mg。在SDS-PAGE上显示出一条蛋白带,其分子量约为37 KD。光谱分析显示,在399nm处有一典型的Soret带,在495nm和636nm处有特征吸收,酶反应的最适pH在5.0左右,最适双氧水浓度为13mM/L,最适温度在40℃左右,有较好的耐热能力。     

Flow injection chemiluminescence determination of sudan I in hot chilli sauce

A chemiluminescence method based on the luminol-H2O2 system with flow injection technology was proposed for the determination of sudan I in hot chilli sauce. It was found that sudan I could enhance chemiluminescence intensity generated from the luminol-H2O2 system. The increment of chemiluminescence intensity was proportional to the concentration of sudan I, giving a calibration graph linear over the concentration from 10 pg mL-1 to 7 ng mL-1 (R 2 = 0.9980) with the detection limit of 3 pg mL-1 (3sigma) and the quantification limit of 7.5 pg mL-1. At a flow rate of 2.0 mL min-1, one analysis cycle, including sampling and washing, could be accomplished in 60 s with a relative standard deviation of <5.0%. The method has been applied successfully to the determination of sudan I in Pixian douban, Golden Mark guilin chilli sauce, and Golden Mark satay sauce, and the recovery was 90.6-110.0%.