Jeanette M. Van Emon

Applications of electrochemical immunosensors to environmental monitoring

This paper discusses basic electrochemical immunoassay technology. Factors limiting the practical application of antibodies to analytical problems are also presented. It addresses the potential use of immunoassay methods based on electrochemical detection for the analysis of environmental samples. It provides examples for the detection and quantitation of environmental samples using conducting electroactive polymers (CEPs). CEP-based immunosensing systems are compared with conventional environmental immunoassay procedures. The advantages of using these types of sensors for rapid, sensitive, and cost-effective analysis of pesticides and toxic chemicals are analysed and discussed. CEP-based immunosensing technology might eventually be used for continuous monitoring of effluents such as waste streams to determine compliance with regulations. CEP-based sensors are suitable for monitoring ground-water, waste stream effluents, agricultural run-offs and for monitoring the effectiveness of remediation, or for other situations where a real-time monitoring capability is desired.

Evaluation of analytical methods for determining pesticides in baby foods and adult duplicate-diet samples

Abstract Determination of pesticides in food is often complicated by the presence of fats and requires multiple clean-up steps before analysis. Cost-effective methods are needed for analyzing the large number of samples generated in large-scale exposure studies. We examined two extraction methods, supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE), coupled with various clean-up techniques for the analysis of pesticides in baby foods and exposure samples. The SFE-gas chromatogram/mass spectrometry (GC/MS) method did not provide quantitative recoveries (

ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum.

Learning ObjectivesRecall the properties of stachylysin and the characteristics of the assays developed to quantify it.Identify the types of indoor environment that harbor Stachybotrys chartarum.Summarize the results of stachylysin assays in rats and humans exposed to S. chartarum. The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.

Immunochemical determination of dioxins in sediment and serum samples

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are considered highly toxic contaminants and the environmental and biological monitoring of these compounds is of great concern. Immunoassays may be used as screening methods to satisfy the growing demand for rapid and low cost analysis. In this work, we describe the application of an immunoassay that uses 2,3,7-trichloro-8-methyldibenzo-p-dioxin (TMDD) as a surrogate standard for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to sediment and human serum samples. Sample extraction and preparation methods were developed with the aim to establish the simplest, cost-effective and efficient removal of the matrix interferences in the enzyme-linked immunosorbent assay (ELISA). The overall method for sediments is based on a hexane extraction; clean up by a multilayered silica gel column and an activated carbon column; an organic solvent exchange with DMSO-Triton X-100 and ELISA measurement. The gas chromatography-high resolution mass spectrometry (GC-HRMS) validation studies (n = 13) revealed that the method is suitable for the toxic equivalents (TEQ) screening of dioxin in sediments with a method detection limit of about 100pgg(-1) dry sediment with a precision of 13-33% R.S.D. The analysis of a large number of samples originating from different sources would be required to establish more precisely the screening level, as well as the number of false positives and negatives of dioxin TEQ by the immunoassay for sediments. The immunoassay method for sediment analysis offers improvement in speed, sample throughput, and cost in comparison to GC-HRMS. Dioxins were determined in serum samples after a simple liquid-liquid extraction and solvent exchange into DMSO-Triton X-100 without further dilution. The current method (approximate method LOQ of 200pgml(-1) serum) is not sufficiently sensitive for the determination of dioxins in serum to measure acceptable exposure limit.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within +/-20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within +/-20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30ng/mL by ELISA and 0.2ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10mL of urine converted into 1mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.

Comparison of immunoassay and gas chromatography–mass spectrometry for measurement of polycyclic aromatic hydrocarbons in contaminated soil

Polycyclic aromatic hydrocarbons (PAHs) are frequently encountered in the environment and may pose health concerns due to their carcinogenicity. A commercial enzyme-linked immunosorbent assay (ELISA), was evaluated as a screening method for monitoring PAHs at contaminated sites. The ELISA was a carcinogenic PAH (C-PAH) RaPID assay testing kit that cross-reacts with several PAHs and utilizes benzo[a]pyrene (BaP) as a calibrator. Soil samples were extracted with 50% acetone in dichloromethane (DCM) for analysis by ELISA and gas chromatography–mass spectrometry (GC–MS). The overall method precision was within ±30% for ELISA and within ±20% for GC–MS. Recovery data for spiked soils ranged from 46 to 140% for BaP as determined by ELISA. Recoveries data of the GC–MS surrogate standards, 2-fluorobiphenyl and chrysene, were greater than 70%. The GC–MS procedure detected a total of 19 priority PAHs (2–6-ring PAHs) including seven probable human carcinogens (4–6-ring B2-PAHs). The ELISA results were compared to GC–MS summation results for the total 19 target PAHs as well as for the subset of the seven B2-PAH compounds. For all soil samples, the PAH concentrations derived from ELISA were greater than the sum of B2-PAH concentrations obtained by GC–MS. ELISA determinations were also frequently greater than the results obtained by GC–MS for the total 19 PAH compounds. This discrepancy can be expected, since the ELISA is a screening assay for the detection of several related PAHs while the GC–MS procedure detects priority PAH compounds. Thus, only a subset of PAHs (e.g. 19 PAHs) in the soil samples were measured by GC–MS while additional PAHs, including alkylated PAHs, and PAH derivatives have been demonstrated to be cross-reactive in the C-PAH ELISA. Results of paired tests show that the PAH data from ELISA and GC–MS methods are significantly different (P<0.001), but highly correlated. The ELISA data had a strong positive relationship with the GC–MS summation data for the B2-PAHs as well as for the 19 PAHs targeted by the GC–MS method. Results indicate that the ELISA may be useful as a broad screen for monitoring PAHs in environmental samples.

Analysis of soil and dust samples for polychlorinated biphenyls by enzyme-linked immunosorbent assay (ELISA)

An inhibition enzyme-linked immunosorbent assay (ELISA) was used to determine polychlorinated biphenyls (PCBs) in house dust and soil. Soil and house dust samples were analyzed for PCB by both gas chromatography/electron capture detection (GC/ECD) and ELISA methods. A correlation coefficient of 0.91 was obtained for the 41 soil samples. The correlation between the GC/ECD and the ELISA methods for the 10 house dust samples was not as good as that for the soil samples.

An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples.

A 96-microwell enzyme-linked immunosorbent assay (ELISA) method was evaluated to determine PCDDs/PCDFs in sediment and soil samples from an EPA Superfund site. Samples were prepared and analyzed by both the ELISA and a gas chromatography/high resolution mass spectrometry (GC/HRMS) method. Comparable method precision, accuracy, and detection level (8 ng kg(-1)) were achieved by the ELISA method with respect to GC/HRMS. However, the extraction and cleanup method developed for the ELISA requires refinement for the soil type that yielded a waxy residue after sample processing. Four types of statistical analyses (Pearson correlation coefficient, paired t-test, nonparametric tests, and McNemars test of association) were performed to determine whether the two methods produced statistically different results. The log-transformed ELISA-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin values and log-transformed GC/HRMS-derived TEQ values were significantly correlated (r=0.79) at the 0.05 level. The median difference in values between ELISA and GC/HRMS was not significant at the 0.05 level. Low false negative and false positive rates (<10%) were observed for the ELISA when compared to the GC/HRMS at 1,000 ng TEQ kg(-1). The findings suggest that immunochemical technology could be a complementary monitoring tool for determining concentrations at the 1,000 ng TEQ kg(-1) action level for contaminated sediment and soil. The ELISA could also be used in an analytical triage approach to screen and rank samples prior to instrumental analysis.

Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence.

Limited by the lack of a sensitive, universal detector, many capillary‐based liquid‐phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid‐phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti‐biotin‐IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser‐induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC‐biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC‐biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).

Evaluation of analytical methods for determining pesticides in baby food

Three extraction methods and two detection techniques for determining pesticides in baby food were evaluated. The extraction techniques examined were supercritical fluid extraction (SFE), enhanced solvent extraction (ESE), and solid phase extraction (SPE). The detection techniques used were enzyme-linked immunosorbent assay (ELISA) and gas chromatography/mass spectrometry (GC/MS). Different SFE and ESE conditions were considered, and the resulting extracts were analyzed by either ELISA or GC/MS. The use of C18 SPE cartridges to extract pesticides from baby food was also evaluated. Using SFE–ELISA, recoveries of most spiked pesticides were less than 50% in both non-fat and fatty baby food. Using SFE–GC/MS, recoveries of target pesticides were greater than 80% in dried baby food, but 10–60% of the spiked pesticides were lost during the freeze-drying process. Off-line coupling of SPE–GC/MS provided a quantitative measure (>80%) of the pesticides in non-fat baby food (fruits and vegetables). Direct ELISA applied to diluted non-fat food also gave a quantitative measure of the target pesticides. The ESE–ELISA method provided a quantitative determination of atrazine in both non-fat and fatty baby food.