Enhancing the Sensitivity of the Bio-barcode Immunoassay for Triazophos Detection Based on Nanoparticles and Droplet Digital Polymerase Chain Reaction

Abstract

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.

Enhanced Bio-Barcode Immunoassay Using Droplet Digital PCR for Multiplex Detection of Organophosphate Pesticides

“…The designed DNA sequences, primers, and probes were amplified on the qPCR instrument to determine whether the target sequences can be amplified. Sequences 1, 2, and 3 were assigned to triazophos, parathion, and chlorpyrifos, respectively, and sequence 1 was selected based on the previous literature . PCR amplified DNA sequences 1, 2, and 3 with probes and primers to determine the specificity of the three groups of sequences.…”

“…The specific procedure was based on our previous report . Briefly, 15 μL of 0.2 mol L –1 K 2 CO 3 solution was added into each of the three glass bottles containing 1 mL of AuNP solution to adjust the pH between 9.0 and 9.5.…”

“…Finally, Stilla made the first commercially available Crystal digital PCR in 2016. The ddPCR can detect a variety of viruses, − pathogenic bacteria, − heavy metals, , and transgenes. − In this context, our group first applied it for the detection of pesticide contaminants . Before the traditional PCR amplification, the reaction systems containing nucleic acid molecules are divided into thousands of microdroplets.…”

Analytical methods to analyze pesticides and herbicides

“…A bio‐barcode competitive immunoassay was established by Cui et al (2019) on the basis of droplet digital polymerase chain reaction (ddPCR) for triazophos analysis. Total recoveries ranged from 76.9% to 94.4%.…”

Aptamer Recognition-Driven Homogeneous Electrochemical Strategy for Simultaneous Analysis of Multiple Pesticides without Interference of Color and Fluorescence

“…In view of this, pesticide residues are hazardous to human health via restraining cholinesterase activity and increasing the amount of acetylcholine existing in biological samples. , Precise determination of the residual level of pesticides in agricultural crops is urgently required. , Unfortunately, traditional chromatographs and mass spectrometers cannot meet this demand because of some undeniable drawbacks caused by large-scale equipment, long operation time, need for trained technicians, and high cost. − Developing ingenious strategies for rapid, sensitive, and reliable detection of pesticides is highly desired. According to our understanding, commonly used approaches are mainly divided into a molecular imprinting technique, − immunoassay, and an enzyme-depression strategy, − which have acquired huge success in the analysis of pesticides, but are governed by some inevitable obstacles. For example, molecularly imprinted polymers are difficult to prepare and are usually used in an organic solvent; immunoassay systems are developed based on high-price antibodies, whose activities are different due to different sources, easily causing poor repeatability; the enzyme-depression strategy cannot selectively differentiate pesticides …”