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Herein, a novel visual method for detecting triazophos based on competitive bio-barcode immunoassay was described. The competitive immunoassay was established by gold nanoparticles (AuNPs), magnetic microparticle (MMPs) and triazophos, combined with biochip hybridization system to detect the residual of triazophos in water and apple. Because AuNPs carried many bio-barcodes, which hybridized with labeled DNA on the biochip, catalyzed signal amplification using silver staining was detected by grayscale values as well as the naked eye. Notably, the grayscale values decreases with increasing the concentrations of triazophos, and the color change weakened gradually. The detection range was in between 0.05 and 10 ng/mL and the minimum detection limit was set at 0.04 ng/mL. Percent recovery calculated from spiked water and apple samples ranged between 55.4 and 107.8% with relative standard deviations (RSDs) of 12.4-24.9%. It has therefore been shown that this protocol provides a new insight for rapid detection of small molecule pesticides in various matrices., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although the toxicity of triazophos is high and it has been pulled from the market in many countries; it is still widely used and frequently detected in agricultural products. While conventional analyses have been routinely used for the quantification and monitoring of triazophos residues, those for detecting low residual levels are deemed necessary. Therefore, we developed a novel and sensitive fluorometric signal amplification immunoassay employing bio-barcodes for the quantitative analysis of triazophos residues in foodstuffs and surface water. Herein, monoclonal antibodies (mAbs) attached to gold nanoparticles (AuNPs) were coated with DNA oligonucleotides (used as a signal generator), and a complementary fluorogenic RNA was used for signal amplification. The system generated detection signals through DNA-RNA hybridization and subsequent dissociation of fluorophores by Ribonuclease H (RNase H). It has to be noted that RNase H can only disintegrate the RNA in DNA-RNA duplex, but not cleave single or double-stranded DNA. Hence, with iterative cycles of DNA-RNA hybridization, sufficient strong signal was obtained for reliable detection of residues. Furthermore, this method enables quantitative detection of triazophos residues through fluorescence intensity measurements. The competitive immunoassay shows a wide linear range of 0.01-100 ng/mL with a limit of detection (LOD) of 0.0032 ng/mL. The assay substantially meets the demand for the low residue detection of triazophos residues in agricultural products and water samples. Accuracy (expressed as spiked recovery %) and coefficient of variation (CV) were ranged from 73.4% to 116% and 7.04% to 17.4%, respectively. The proposed bio-barcodes immunoassay has the advantages of being stable, reproducible, and reliable for residue detection. In sum, the present study provides a novel approach for detection of small molecules in various sample matrices., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An ultrasensitive bio-barcode competitive immunoassay method based on droplet digital polymerase chain reaction (ddPCR) was developed for the determination of triazophos. Gold nanoparticles (AuNPs) were coated with monoclonal antibodies (mAbs) and complementary double-stranded DNA (dsDNA), which included bio-barcode DNA and thiol-capped DNA. Magnetic nanoparticle (MNP) probes were constructed by modifying the MNPs with ovalbumin-hapten conjugates (OVA-hapten). The target pesticide and OVA-hapten on the surface of the MNP probes competed with the AuNP probes simultaneously, and then the bio-barcode DNA was released for quantification by ddPCR. The concentration of released DNA was inversely proportional to the concentration of pesticide to be tested. Under the optimum conditions, the competitive immunoassay exhibited a wide linear range of 0.01-20 ng/mL and a low detection limit of 0.002 ng/mL. Spike recovery tests were carried out using apple, rice, cabbage, and cucumber samples to verify the feasibility of the method. The recovery and relative standard deviations (RSDs) of the technique ranged from 76.9 to 94.4% and from 10.8 to 19.9%, respectively. To further validate the results, a linear correlation analysis was performed between the proposed method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Consequently, the bio-barcode immunoassay based on nanoparticles and ddPCR, an ultrasensitive method, showed great potential for the determination of target pesticides in real samples.

A simple and highly sensitive immunoassay based on a competitive binding and bio-barcode amplification was designed for detection of small molecules, triazophos. The gold nanoparticles (AuNPs) were modified with monoclonal antibodies and 6-carboxyfluorescein labeled single-stranded thiol-oligonucleotides (6-FAM-SH-ssDNAs); the fluorescence of 6-FAM was quenched by AuNPs. Ovalbumin-linked haptens were coated on the bottom of microplate to compete with the triazophos in the sample for binding to the antibodies on the AuNP probes. The fluorescence intensity was inversely proportional to analyte concentration. Parameters of AuNP probes preparation and immune reaction were optimized. At the optimal conditions, the salting process was shortened to 1 h and 166 ± 9 ssDNAs were loaded onto a single AuNP. The competitive fluorescence bio-barcode immunoassay was performed on water, rice, cucumber, cabbage and apple samples. The linear range of the method was 0.01-20 μg L -1 and the limit of detection (LOD) was 6 ng L -1 . The recovery and relative standard deviations (RSDs) ranged from 85.0 to 110.3% and 9.4-17.4%, respectively. Good correlations were obtained between the results of the developed method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, it is suggested that the competitive fluorescent bio-barcode immunoassay had the potential to be used as a sensitive method for detection of a variety of small molecules in various samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel detection method of small molecules, competitive bio-barcode amplification immunoassay, was developed and described in this report. Through the gold nanoparticles (AuNPs) probe and magnetic nanoparticles (MNPs) probe we prepared, only one monoclonal antibody can be used to detect small molecules. The competitive bio-barcode amplification immunoassay overcomes the obstacle that the bio-barcode assay cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. The small molecular compounds, triazophos, were selected as targets for the competitive bio-barcode amplification immunoassay. The linear range of detection was from 0.04 ng mL -1 to 10 ng mL -1 , and the limit of detection (LOD) was 0.02 ng mL -1 , which was 10-20 folds lower than ELISA (Enzyme Linked Immunosorbent Assay). A practical application of the proposed immunoassay was evaluated by detecting triazophos in real samples. The recovery rate ranged from 72.5% to 110.5%, and the RSD was less than 20%. These results were validated by GC-MS, which indicated that this convenient and sensitive method has great potential for small molecular in real samples.

Herein, a novel visual method for detecting triazophos based on competitive bio-barcode immunoassay was described. The competitive immunoassay was established by gold nanoparticles (AuNPs), magnetic microparticle (MMPs) and triazophos, combined with biochip hybridization system to detect the residual of triazophos in water and apple. Because AuNPs carried many bio-barcodes, which hybridized with labeled DNA on the biochip, catalyzed signal amplification using silver staining was detected by grayscale values as well as the naked eye. Notably, the grayscale values decreases with increasing the concentrations of triazophos, and the color change weakened gradually. The detection range was in between 0.05 and 10 ng/mL and the minimum detection limit was set at 0.04 ng/mL. Percent recovery calculated from spiked water and apple samples ranged between 55.4 and 107.8% with relative standard deviations (RSDs) of 12.4-24.9%. It has therefore been shown that this protocol provides a new insight for rapid detection of small molecule pesticides in various matrices.

Although the toxicity of triazophos is high and it has been pulled from the market in many countries; it is still widely used and frequently detected in agricultural products. While conventional analyses have been routinely used for the quantification and monitoring of triazophos residues, those for detecting low residual levels are deemed necessary. Therefore, we developed a novel and sensitive fluorometric signal amplification immunoassay employing bio-barcodes for the quantitative analysis of triazophos residues in foodstuffs and surface water. Herein, monoclonal antibodies (mAbs) attached to gold nanoparticles (AuNPs) were coated with DNA oligonucleotides (used as a signal generator), and a complementary fluorogenic RNA was used for signal amplification. The system generated detection signals through DNA-RNA hybridization and subsequent dissociation of fluorophores by Ribonuclease H (RNase H). It has to be noted that RNase H can only disintegrate the RNA in DNA-RNA duplex, but not cleave single or double-stranded DNA. Hence, with iterative cycles of DNA-RNA hybridization, sufficient strong signal was obtained for reliable detection of residues. Furthermore, this method enables quantitative detection of triazophos residues through fluorescence intensity measurements. The competitive immunoassay shows a wide linear range of 0.01-100 ng/mL with a limit of detection (LOD) of 0.0032 ng/mL. The assay substantially meets the demand for the low residue detection of triazophos residues in agricultural products and water samples. Accuracy (expressed as spiked recovery %) and coefficient of variation (CV) were ranged from 73.4% to 116% and 7.04% to 17.4%, respectively. The proposed bio-barcodes immunoassay has the advantages of being stable, reproducible, and reliable for residue detection. In sum, the present study provides a novel approach for detection of small molecules in various sample matrices.

A rapid, ultra-sensitive and simple fluorescence immunoassay (FLISA) on CdSe/ZnS Quantum dots (QDs) for recognition pesticide triazophos have been established. In this assay, the immunoassay uses (CdSe/ZnS) Quantum dots (QDs) as a probe that carries monoclonal antibody (mAb). The free triazophos and OVA-haptens were competed for binding the mAb on the surface of the QDs. Therefore, the concentration of TRIAZ can get by calculation of the detection of fluorescence value. The assay has a linear response in the 10 ng L−1–25 μg L−1 TRIAZ concentration range, and limit of detection (IC10) was 0.508 ng L−1. The recovery rate in the case of spiked samples ranges from 82.6% to 96.6%, and the RSD is <20%. FLISA can accurately and sensitively screen out triazophos in representative actual samples that contain trace levels of pesticides, it can play a prominent part in agricultural product and environmental detection. [ABSTRACT FROM AUTHOR]

The small molecule compounds like pesticide, veterinary drug, bio-toxin, and heavy metal are widely found in animals, plants, soil, etc. Excessive compounds residues will have a bad influence on human health and the environment. Thus, it is extremely urgent that can detect the small molecules simultaneously. At present, many researches of simultaneous detection for small molecules using the method of an immunoassays have been reported thanks to its advantages of fast speed, simple operation, and high specificity. The small molecules have only one antigenic determinant, so the competitive immunoassay is the main method for small molecule compounds detection. In this paper, our main job is to describe the development of immunoassay for multi-residue detection of small molecule compounds and introduce three ways to complete the analysis of multi-residue immunoassay of small molecule compounds. We also summarize deficiencies and make an expectation of the immunoassays. [ABSTRACT FROM AUTHOR]

This study reports a novel bio-bar-code amplification competitive immunoassay for the detection of pesticide residue combined with a micro well plate. The sensing competitive immunoassay was made up of two types of particles: (1) the gold nanoparticles (AuNPs) with recognition elements (monoclonal antibody: McAb) and hundreds of thiolated single-strand oligonucleotide bar-codes; and (2) the magnetic microparticle (MMPs) with competition elements (hapten-OVA) for the target pesticide. Target pesticide and pesticide hapten competed with McAb labeled on the surface of AuNPs. The bar-code strands were released by dithiothreitol. Then a simple method based on micro well plate was used for the quantification of the bar-code strands. The bar-code strands with the half-complementary biotinylated DNA and half-complementary DNA-modified gold nanoparticles probe were immobilized onto the surface of a streptavidin-coated micro well plate, and the signal was amplified by silver enhancement. Triazophos (TAP), as a pesticide model, was detected by the proposed immunoassay. The linear range was from 2.5 × 10 −2 to 40.0 ng mL −1 , and LOD of the immunoassay was 1.96 × 10 −2 ng mL −1 . The correlation was further studied between this method and GC–MS analysis. Therefore, this method provided a promising approach for rapid detection and screening of pesticide residue. [ABSTRACT FROM AUTHOR]