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A novel antibody-based biosensor method for the rapid measurement of PAH contamination in oysters.

ABSTRACT: Conventional PAH analytical methods are time-consuming and expensive, limiting their utility in time sensitive events (i.e. oil spills and floods) or for widespread environmental monitoring. Unreliable and inefficient screening methods intended to prioritize samples for more extensive analyses exacerbate the issue. Antibody-based biosensor technology was implemented as a quantitative screening method to measure total PAH concentration in adult oysters (Crassostrea virginica) - a well-known bioindicator species with ecological and commercial significance. Individual oysters were analyzed throughout the historically polluted Elizabeth River watershed (Virginia, USA). Significant positive association was observed between biosensor and GC-MS measurements that persisted when the method was calibrated for different regulatory subsets of PAHs. Mapping of PAH concentrations in oysters throughout the watershed demonstrates the utility of this technology for environmental monitoring. Through a novel extension of equilibrium partitioning, biosensor technology shows promise as a cost-effective analysis to rapidly predict whole animal exposure to better assess human health risk as well as improve monitoring efforts.

SUBMITTER: Prossner KM

PROVIDER: S-EPMC9531917 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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A novel antibody-based biosensor method for the rapid measurement of PAH contamination in oysters.

Prossner Kristen M KM Vadas George G GG Harvey Ellen E Unger Michael A MA

Environmental technology & innovation 20220421

Conventional PAH analytical methods are time-consuming and expensive, limiting their utility in time sensitive events (i.e. oil spills and floods) or for widespread environmental monitoring. Unreliable and inefficient screening methods intended to prioritize samples for more extensive analyses exacerbate the issue. Antibody-based biosensor technology was implemented as a quantitative screening method to measure total PAH concentration in adult oysters (<i>Crassostrea virginica</i>) - a well-know ...[more]

PMID: 36204483

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Project description:The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Project description:BackgroundSalicylic acid (SA) is an important signalling molecule in plant defenses against biotrophic pathogens. It is also involved in several other processes such as heat production, flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate (salicylic acid 2-O-beta-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_lux. In this paper we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.ResultsOn the basis of the original biosensor-based method, we optimized extraction and quantification. SAG content was determined by treating crude extracts with beta-glucosidase, then measuring the released free SA with the biosensor. beta-glucosidase treatment released more SA in acetate buffer extract than in Luria-Bertani (LB) extract, while enzymatic hydrolysis in either solution released more free SA than acid hydrolysis. The biosensor-based method detected higher amounts of SA in pathogen-infected plants than did a GC/MS-based method. SA quantification of control and pathogen-treated wild-type and sid2 (SA induction-deficient) plants demonstrated the efficacy of the method described. Using the methods detailed here, we were able to detect as little as 0.28 mug SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.ConclusionThe ability of Acinetobacter sp. ADPWH_lux to detect SA in a complex mixture, combined with the enzymatic hydrolysis of SAG in crude extract, allowed the development of a simple, rapid, and inexpensive method to simultaneously measure free and glucose-conjugated SA. This approach is amenable to a high-throughput format, which would further reduce the cost and time required for biosensor-based SA quantification. Possible applications of this approach include characterization of enzymes involved in SA metabolism, analysis of temporal changes in SA levels, and isolation of mutants with aberrant SA accumulation.

Dataset Information

A novel antibody-based biosensor method for the rapid measurement of PAH contamination in oysters.

Publications

A novel antibody-based biosensor method for the rapid measurement of PAH contamination in oysters.

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