A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl.

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15 nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody-DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5 pg L(-1) to 10 ng L(-1), and the limit of detection (LOD) was 1.72 pg L(-1). Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82 % to 112 %, and the coefficient of variation (CV) was acceptable. The results were compared with GC-ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

Development of a new polyclonal antibody for the determination of polychlorinated biphenyls in indoor air by ic-ELISA.

A new polyclonal antibody (pAb) was prepared and used for the determination of polychlorinated biphenyls (PCBs) in air samples to promote the application of immunoassay technology in the determination of PCBs. Three PCB congeners immunogen mixture was used to stimulate immune responses in rabbits. The specific pAb to PCBs was obtained and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). A standard curve for Aroclor 1248 was prepared using concentrations ranging from 0.1 to 100 µg L(-1). The average IC50 value was 16.21 µg L(-1) and the limit of detection at 10% inhibition (IC90) was 0.069 µg L(-1). The entire procedure was then evaluated using spiked air samples. The recoveries of Aroclor 1248 at various spiking levels in the air samples ranged from 84 to 113%, with relative standard deviations of 3 to 6%. Under optimum conditions, the cross-reactivity profiles of the assays were obtained using three selected congeners, four Aroclor products, and other structurally related compounds of PCBs. The assays were found to be highly specific for PCB congeners and Aroclors 1248 and 1242. The air samples were then analyzed using gas chromatography coupled with high-resolution mass spectrometry to confirm the ic-ELISA results. The attained results demonstrated that the proposed method was an effective and inexpensive technique for the PCBs determination in air samples.

Real-time immuno-PCR assay for detecting PCBs in soil samples.

A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37 hapten-ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml(-1) antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method, and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds in the environment.

Determination of anthracene by real-time immuno-polymerase chain reaction assay.

A reliable and sensitive competitive real-time immuno-PCR (RT-IPCR) assay for the determination of anthracene (AN) was developed. 9-Anthracenebutanoic acid, gamma-oxo-(ANA) was synthesized as the hapten of AN. Mixed anhydride reaction (MAR) was used to couple the ANA to ovalbumin (OVA) to form artificial coating antigen. Active ester method (AEM) was used to couple the ANA to bovine serum albumin (BSA) to form artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel RT-IPCR assay for determination of AN was described. Under optimized assay conditions, AN can be determined in the concentration range from 1 fgmL(-1) to 100 pgmL(-1) with a detection limit of 0.5 fgmL(-1). The cross-reactivities of the anti-AN antibody to seven structurally related compounds were below 15%. Environmental water samples were successfully analyzed, showing a good accuracy and suitability to analyze AN in field samples. Recovery was between 93.3% and 120.0% and would be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.

Determination of naphthalene by competitive fluorescence immunoassay.

A reliable and sensitive competitive fluorescence immunoassay for the quantitative determination of naphthalene (NA) was developed. 2-naphthoxy acetic acid (NAA) was selected as the hapten of naphthalene. Active ester method (AEM) was used to couple the NAA to carrier proteins (bovine serum albumin) to form artificial immune antigen. Male New Zealand white rabbits were immunized with this antigen to obtain polyclonal antibodies, with which, a novel fluorescence immunoassay for detection of NA was described. Under best conditions, NA can be determined in the concentration range of 0.1-100 microg/L with a detection limit of 0.05 microg/L. The cross-reactivities of the anti-NA antibody to seven structurally related compounds were below 15%. Some environmental samples were analyzed with satisfactory results. It shows a good accuracy and suitability to analyze NA in environmental water.

Detection of trace anthracene in soil samples with real-time fluorescence quantitative immuno-PCR using a molecular beacon probe.

We developed a highly sensitive and robust real-time fluorescence quantitative immuno-PCR (RTFQ-IPCR) method which uses molecular beacon (MB) probe to detect trace anthracene in the environment. This method was performed on serial dilutions of known anthracene concentrations equivalent to 10-fold dilutions of 10fg/mL to 100pg/mL. We obtained a linear relationship between 10fg/mL and 100pg/mL, with y=0.684x+13.221. A correlation coefficient of 0.994 was also identified, with a detection limit of 4.5fg/mL. After investigating the presence of anthracene in soil samples via RTFQ-IPCR, the obtained concentrations were confirmed by ELISA to be correct and believable, with the recovery ratio ranging from 82% to 112.5%. Based on its sensitivity and reproducibility, MB-based RTFQ-IPCR was found to be acceptable for use in on-site field tests to provide rapid, quantitative, and reliable test results for making environmental decisions.

Determination of phenanthrene by antibody-coated competitive real-time immuno-PCR assay.

A reliable selective and sensitive antibody-coated competitive real-time immuno-PCR (RT-IPCR) assay for the determination of phenanthrene (PH) was developed. Phenanthrene butanoic acid (gamma-oxo-PHA) was synthesized as the hapten of PH. An active ester method was used to couple the PHA to bovine serum albumin to form an artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies, with which a novel RT-IPCR assay for determination of PH was developed. Under the optimized assay conditions, PH can be determined in the concentration range from 10 fg/mL to 100 pg/mL with a detection limit of 5 fg/mL. The cross-reactivities of the anti-PH antibody to seven structurally related compounds were below 12.5%. Some environmental water samples were analyzed with satisfactory results, which showed good accuracy and suitability to analyze PH in environmental water. Compared with high-performance liquid chromatography, the recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.

Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay.

Chinese toad, Bufo bufo gargarizans, is frequently found in rice fields, muddy ponds, wetlands and other aquatic ecosystems in China. Because of its habitat, it has many chances of being exposed to pesticides, such as acetochlor, butachlor, chlorimuron-ethyl, and paraquat, which are extensively used in rice or cereal fields. Amphibians may serve as model organisms for determining the genotoxic effects of pollutants contaminating these areas. In the present study DNA damage was evaluated in the Chinese toad using the comet assay, as a potential tool for the assessment of ecogenotoxicity. The first step was to determine the acute toxicity of the above-mentioned herbicides. In acute tests, tadpoles were exposed to a series of relatively high concentrations of acetochlor, butachlor, chlorimuron-ethyl, and paraquat for 96 h. The LC(50 )(96 h) of acetochlor, butachlor, chlorimuron-ethyl and paraquat were measured as 0.76, 1.32, 20.1 and 164 mg l(-1), respectively. Also, negative effects on the behavior of tadpoles were observed with acetochlor, butachlor, and paraquat. Secondly, the comet assay was used for detecting DNA damage in Chinese toad tadpoles exposed to sublethal concentrations of four herbicides. Significant (P < 0.05) concentration-dependent increase in DNA damage (as indicated by tail length, tail moment, olive tail moment) were observed from erythrocytes of tadpoles exposed to sublethal concentrations of acetochlor, butachlor, paraquat, and methyl methanesulfonate, except chlorimuron-ethyl. To our knowledge, this is the first report describing the use of Bufo bufo gargarizans for genotoxicity assessment of herbicides.

[Simultaneous determination of 1-naphthol and 2-naphthol in water by spectrofluorimetry].

A simple and sensitive method for the simultaneous determination of trace amounts of 1-naphthol and 2-naphthol by fluorescence spectrofluorimetry was established based on the fact that the excitation and emission wavelengths are different between 1-naphthol and 2-naphthol. The influencing factors, such as pH temperature and surface activator were synthetically considered in the present paper. Interference of similar organic matters and inorganic ions were also taken into account. Compared with traditional methods, the method discussed here needs no complex pretreatments and expensive equipment. So it is convenient, quick and cheap yet with a satisfactory precision and accuracy. The accuracy and precision of results obtained are satisfactory when the concentration ratio (CR) of 1-naphthol to 2-naphthol is in the range of 1:19-19:1; the detection limit (DL) of both naphthols is 8.6 x 10(-3) ng x mL(-1); the relative standard deviation (RSD) of 1-naphthol and 2-naphthol is 1.26% and 1.45% respectively; and the recovery ratio (RR) of both naphthols is between 95% and 105%. This proposed method has been applied to the determination of trace amounts of 1-naphthol and 2-naphthol in the man-made sample water and environmental imitative water simultaneously, and obtained a satisfactory result, showing that the method discussed here is simple, sensitive and reliable in the future use.

Photocatalytic degradation of Acid Blue 62 over CuO-SnO2 nanocomposite photocatalyst under simulated sunlight.

The novel CuO-SnO2 nanocomposite oxide photocatalysts were prepared by simple co-precipitation method, and characterized by X-ray diffraction, transmission electron microscopy, N2 adsorption-desorption measurement and UV-Vis diffuse reflectance spectroscopy. The photocatalytic activities of CuO-SnO2, evaluated using the photodegradation of Acid Blue 62 as a probe reaction under the irradiation of Xenon light, were also found to be related to the calcination temperature and the molar ratio of Cu to Sn. The maximum photocatalytic activity of the CuO-SnO2 photocatalyst was observed to be calcined at 500 degrees C for 3 h (the molar ratio of Cu to Sn was 1:1) due to the sample with good crystallization and high surface area. It also showed much higher photocatalytic activity in treatment dye wastewater under simulated sunlight irradiation compared to Degussa P25 TiO2.

[Study of dicyclohexyl phthalate on preparation and characterization of artificial antigen].

OBJECTIVE: To study the preparation and characterization of hapten and artificial antigen of dicyclohexyl phthalate. METHODS: A dicyclohexyl phthalate hapten (4-amino dicyclohexyl phthalate) was synthesized by introducing amino as a substituent on the aromatic ring and retaining the ester group, and characterized by 1HNMR, IR and UV. The hapten was conjugated to BSA via amino diazotization linkage. RESULTS: Lambda1 = 214nm, lambda2 = 256nm for the UV of dicyclohexyl 4-nitrophthalate and lambda1 = 226nm, lambda2 = 288nm for the UV of dicyclohexyl 4-aminophthalate. Artificial antigen was prepared and tested by fluorescence, and lambda(ex) = 307nm, lambda(em) = 468nm, and the approximate molar ratio of dicyclohexyl 4-aminophthalate to BSA was 19. The product was used as an immunogen, demonstrating that it is suitable for polyclonal antibody production. CONCLUSION: It is a good method for preparation of artificial antigen of dicyclohexyl phthalate by introducing amino as a substituent on the aromatic ring and retaining the ester group. It was suggested that could supply excellent immune antigen for further preparation of antibody and immunoassay to dicyclohexyl phthalate.

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate.

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 microg L(-1), a dynamic range of approximately 0.1-300 microg L(-1). Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.

[Induction of free radicals after dietary exposure by mercury contaminated rice and protective effect of coexisting selenium].

Sprague-Dawley rats were reared by environmental mercury contaminated rice to survey the potential health risk of Wanshan mercury mining area. Electron spin resonance (ESR) was introduced to detect the species and the intensities of free radicals, using spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that the mercury-contaminated rice significantly increased the levels of free radicals and MDA in rat brain at 7 days (p < 0.05). ESR spectrums showed that the principal spin adducts resulted from the trapping of alkyl free radical (alphaH = 22.7 x 10(-4)T +/- 1.6 x 10(-4)T, alphaN = 15.5 x 10(-4)T +/- 0.5 x 10(-4)T), and hydroxyl radical. Levels of free radicals and MDA increased slowly until after 90-day exposure period (83%, 100%). Element correlation analysis showed high correlations of mercury and selenium in the brain of rat fed with Wanshan rice, suggesting that the coexisting selenium in rice exhibited antagonistic effects on both mercury accumulation and toxicity. The slight increases of free radicals in rat brain at 7, 20 and 30-day exposure periods should be related with the scavenger effect of Se.

[Determination of dopamine hydrochloride by a reverse flow injection chemiluminescence method].

A novel chemiluminescence system coupled with a reverse flow injection analysis for the determination of dopamine hydrochloride was presented. It is based on th e strong quench effect of dopamine hydrochloride on the chemiluminescence reaction between luminol and hexacyanoferrate(III) under alkaline condition. Various factors affecting the chemiluminescence intensity of the system were investigated. The possible mechanism of the proposed method was also studied. The decrease of chemiluminescence intensity was linear with the dopamine hydrochloride content in the range of 2.0 x 10(-9) -8.0 x 10(-7) g x mL(-1), the detection limit of the method was 1.14 x 10(-9) g x mL(-1), and the relative standard deviation was 0.99% (4.0 x 10(-7) g x mL(-1), n = 11). It was successfully used for the determination of the content of dopamine hydrochloride in dopamine hydrochloride injection.

[Flow injection chemiluminescence method for the determination of phenol with N-bromosuccinimide-luminol system].

A novel flow injection chemiluminescence method was presented for the determination of phenol. The method is based on the quenching effect of phenols on the chemiluminescence reaction between luminol and N-bromosuccinimide. The linear range for the determination of phenol is 1.0 x 10(-5) -9.0 x 10(-4) mg x mL(-1), and the detection limit is 1.81 x 10(-7) mg x mL(-1). This method was used for the determination of phenol in water samples with satisfactory results. The mechanism of the reaction was also expounded.

[Study on the relationships between chemiluminescent properties and molecular structures of some biacridine probes].

The chemiluminescent (CL) properties of biacridine probes, such as 10,10'-dimethyl-3,3'-disulfo-9,9'-biacridine (DMDSBA), 3,3'-disulfo-9,9'-biacridine dinitrate (DSBADN), 10,10'-diethcarboxyl-9,9'-biacridine dinitrate (DEBADN), 10,10'-dimethyl-3,3'-diamino-9,9'-biacridine (DMDABA), and 10,10'-di(4-aminobutyl)-9,9-biacridine dinitrate (DABADN), were studied in this paper, and the relationships between properties and molecular structures were obtained. A new theory and some valuable experimental data have been provided for the investigation and exploitation of the new CL probes for biomacromolecules.

[Study on the chemiluminescence (CL) analysis for the determination of pyrogallol in water environment].

The effect of polyhydroxyphenols on the new chemiluminescence (CL) system of isothiocyanatoisoluminol (ITCI) was studied. Pyrogallol was found to enhance the CL obviously, which provided a new CL method for the determination of pyrogallol. Effect of pH, KIO4 and ITCI concentration on the CL intensity of the system was investigated. An intensive interference study of usual ions and screen reagent was also carried out. Under optimum conditions, a linear relationship between pyrogallol and CL intensity was observed in the range of 1.75 x 10(-8) -2.5 x 10(-6) mol x L(-1). The detection limit of pyrogallol was 3.49 x 10(-9) mol x L(-1) (DL = 3 s/r). The relative standard deviation was 5.60% (5 x 10(-7) mol x L(-1) pyrogallol, n = 9). Furthermore, the method has been applied to the detection of pyrogallol in a simulated sample with satisfactory results. The recovery of the method was 94.0%-112.0%.