Guomin Shan

Development of toxicity identification evaluation procedures for pyrethroid detection using esterase activity

Recent agrochemical usage patterns suggest that the use of organophosphate (OP) pesticides will decrease, resulting in a concomitant increase in pyrethroid usage. Pyrethroids are known for their potential toxicity to aquatic invertebrates and many fish species. Current toxicity identification evaluation (TIE) techniques are able to detect OPs, but have not been optimized for pyrethroids. Organophosphate identification methods depend upon the use of piperonyl butoxide (PBO) to identify OP-induced toxicity. However, the use of PBO in TIE assays will be confounded by the co-occurrence of OPs and pyrethroids in receiving waters. It is necessary, therefore, to develop new TIE procedures for pyrethroids. This study evaluated the use of a pyrethroid-specific antibody, PBO, and carboxylesterase activity to identify pyrethroid toxicity in aquatic toxicity testing with Ceriodaphnia dubia. The antibody caused significant mortality to the C. dubia. Piperonyl butoxide synergized pyrethroid-associated toxicity, but this effect may be difficult to interpret in the presence of OPs and pyrethroids. Carboxylesterase activity removed pyrethroid-associated toxicity in a dose-dependent manner and did not compromise OP toxicity, suggesting that carboxylesterase treatment will not interfere with TIE OP detection methods. These results indicate that the addition of carboxylesterase to TIE procedures can be used to detect pyrethroids in aquatic samples.

Highly sensitive dioxin immunoassay and its application to soil and biota samples

Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known highly toxic compound that is present in nearly all components of the global ecosystem, including air, soil, sediment, fish and humans. Dioxin analysis is equipment intensive and expensive requiring low ppt or even ppq levels of detection. A simple, rapid, cost-effective method of analysis is desired to enable researchers to explore issues involving dioxin more quickly and to make more rational regulatory decisions. A sensitive immunoassay for TCDD has been developed in this laboratory. In the present study, this assay is further optimized using a new coating antigen system and validated with biota samples by HRGC-HRMS. The I50 of current assay to 2,3,7-trichloro, 8-methyldibenzo-p-dioxin (TMDD), a surrogate standard for TCDD, is 36 ± 6.0 ppt, and the lower detection limit (LDL) is 4 ppt in the buffer. A good agreement between immunoassay and GC/MS was achieved for fish and egg samples. To interface with the immunoassay, a rapid sample preparation method was developed for soil samples. Without any further cleanup, the soil extracts can be directly used in the ELISA with a detection limit at the low ppt level. It can be used as an on-site tool for environmental monitoring.

Development of a class-specific immunoassay for the type I pyrethroid insecticides

A general enzyme-linked immunosorbent assay was developed for the type I pyrethroid insecticides, such as permethrin, phenothrin, resmethrin and bioresmethrin. Polyclonal antibodies were generated by immunizing with a permethrin derivative, 2,2-dimethyl-3-(5 � -carboxy-pent-1 � -en-yl)cyclopropanecarboxylic acid-(3-phenoxybenzyl)ester conjugated with thyroglobulin, bovine serum albumin or ovalbumin. Antisera were screened against eight different coating antigens. The antibody–antigen combination with the lowest background, and highest sensitivity for permethrin was further optimized and tested for solvent and detergent tolerance. The I50’s of the optimized immunoassay were 30g/l for permethrin and 20g/l for phenothrin, respectively. No cross-reactivities were measured to the type II pyrethroids, such as esfenvalerate, cyfluthrin, cypermethrin, deltamethrin, fenvalerate and fluvalinate. This assay can be used in monitoring studies to distinguish between types I and II pyrethroids.

Development of dioxin toxicity evaluation method in human milk by enzyme-linked immunosorbent assay: assay validation for human milk

In this study, the development of a toxicity evaluation method for dioxins in human milk by enzyme-linked immunosorbent assay (ELISA) was reported. A total of 17 human milk samples were tested by ELISA and by gas chromatography/mass spectrometry (GC/MS) to assess whether the ELISA performed on samples obtained from primiparas could be considered as reliable enough for identifying a dioxins contamination in human milk. The concept of toxicity equivalent quantity (TEQ) screening was validated by comparing TEQ values for a set of human milk samples to the ELISA responses predicted for those samples. A fairly good correlation (r = 0.920) between immunoassay and GC/MS was achieved for human milk. This ELISA should be useful for biological samples monitoring.

Homogeneous fluoroimmunoassay of a pyrethroid metabolite in urine

Pyrethroids are widely used in agriculture as insecticides. In this study, we describe a simple one-step homogeneous fluoroimmunoassay for the glycine conjugate of phenoxybenzoic acid (PBAG), a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Quenching fluoroimmunoassay (QFIA) is based on the competition of labeled and non-labeled pesticide for binding with antibodies and the resulting calibration curve is based on the relationship between analyte concentration and fluorescence quenching of labeled pesticide by specific antibodies. We developed a QFIA for PBAG in aqueous solution using fluorescein-labeled PBAG and polyclonal antibodies. The estimated IC50 (analyte concentration giving 50% inhibition of quenching) for PBAG was 4.5 nM. The detection limit (DL) was 0.9 nM. The dynamic range of the calibration curve was 2–50 nM. The average analytical recovery obtained by applying the method to urine samples (400or 1000-fold urine dilution) was 85–111%. This demonstrates the QFIA to be a very simple and rapid detection method for PBAG; no washing steps and no enzyme conjugates were required.

Fluorescence quenching competitive immunoassay in micro droplets.

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-microm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 x 512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen-antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay.

Design, synthesis and evaluation of novel P450 fluorescent probes bearing α-cyanoether

Abstract Four α-cyano-containing ethers based on 2-alkoxy-2-naphthylacetonitriles have been designed as a novel structural class of cytochrome P450 fluorescent probes. Their syntheses, fluorescence properties and evaluation in the fluorogenic assay of cytochrome P450 monooxygenase are reported. After P450 enzymatic O -dealkylation, the cyanohydrin metabolite of the four new substrates rearranges to a fluorescent aromatic aldehyde with a larger Stokes shift, and the new substrates exhibit higher specific activities than that of the commercial substrate 7-ethoxyresorufin (ER).

Comparison of immunoaffinity column recovery patterns of polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans on columns generated with different monoclonal antibody clones and polyclonal antibodies

Evaluation of immunoaffinity columns (IACs) for dioxin serum sample clean-up requires a determination of the recovery of various dioxin congeners. We compared the IAC performance of different monoclonal and polyclonal anti-dioxin antibodies, measuring the recovery of congeners of polychlorinated dibenzo-p-dioxins (PCDDs). In addition, we measured the recovery of congeners of the structurally related polychlorinated dibenzofurans (PCDFs). The polyclonal antibody based IACs evaluated had lower recovery for highly chlorinated dioxin congeners, but were more specific toward 2,3,7,8-TCDD. The resemblance of the hapten to 2,3,7,8-TCDD appeared to play a clear role, but chlorines in the 2-, 3- and 7-positions of the hapten were essential. Recovery of 2,3,7,8-TCDD from the IAC showed some relation to the affinity for the antibody measured by either the Ka from accelerator mass spectrometry (AMS) or with 50% inhibition of color activity (IC50) determined from an ELISA analysis. The IACs prepared from four monoclonal antibodies (Mabs) derived from a common hapten showed differences in their retention patterns of PCDDs/PCDFs. Comparison of IC50 from ELISA with recovery from the IACs indicated that a minimum IC50 of 100 ppb was required for satisfactory recovery from the IAC, but the correlation was poor, indicating other factors were involved. Mab DD3 showed the broadest spectrum of the Mabs and showed satisfactory recoveries of all of the dioxin congeners, except OCDD. In addition, DD3 showed good recovery toward 2,3,4,8-TCDF, 2,3,4,7,8-PeCDF and 2,3,4,6,7,8-HxCDF but has poor recovery when PCDFs have a chlorine substitution in the 1-position.

C 60 nanoparticle quenching used as a biological label

Carbon 60 has been used in a functionalized form in a bioassay for a common herbicide, atrazine. It was found that the C60 is a very effective quencher of fluorescence from a number of common dyes. C60 was conjugated to atrazine for use in an immunoassay in which fluorescence from rhodamine was measured. Quenching of the rhodamine emission provided a detection scheme in this assay that yielded a very good limit of detection. The C60 quenching scheme can be used with a wide variety of fluorescent dyes, permitting the potential use of a range of small, cheap excitation sources.

Fluorescence quenching competitive fluoroimmunoassay in microdroplets

The use of micro droplets as a medium for sensitive detection in fluorescence-based immunoassays has been explored in two contexts. The competitive immuno-reaction of a pesticide hapten, esfenvalerate, with its antibody was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-micrometers diameter orifice. Fluorescence from Rhodamine 6G was excited by the second harmonic of a Nd:YAG laser and detected by a 1/8 m imaging spectrograph with a 512 X 512 thermoelectrically cooled, charged-coupled device (CCD) camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased due to quenching. When a sample of esfenvalerate was added to the droplets, esfenvalerate and esfenvalerate- rhodamine conjugate competed for binding with the anti- esfenvalerate antibody. The release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. The assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. Micro droplets also exhibit strong cavity-dependent optical behavior that gives rise to lasing action. Lasing from Rhodamine fluorescence was quenched by the addition of a second dye, oxonol, that absorbs in the spectral region where the Rh 6G fluorescence peaked. A very strong impact on the droplet resonances was observed, leading to the possible use of quenching as an assay for oxonol-labeled haptens.