A novel sustainable immunoassay for sensitive detection of atrazine based on the anti-idiotypic nanobody and recombinant full-length antibody.

Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC50) of 1.66 ng mL-1 for ATR and a linear range of 0.35-8.73 ng mL-1. The cross-reactivity of the assay to ametryn was 64.24%, whereas that to terbutylazine was 38.20%. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40-105.36% for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water.

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 µg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Immunoassays and Emerging Analytical Techniques of Fipronil and its Metabolites for Food Safety: A Review.

Fipronil, classified as a phenylpyrazole insecticide, is utilized to control agricultural, public health, and veterinary pests. Notably, its unique ecological fate involves degradation to toxic metabolites, which poses the risk of contamination in water and foodstuffs and potential human exposure through the food chain. In response to these concerns, there is a pressing need to develop analytical methodologies for detecting fipronil and its metabolites. This review provides a concise overview of the mode of action, metabolism, and toxicology of fipronil. Additionally, various detection strategies, encompassing antibody-based immunoassays and emerging analytical techniques, such as fluorescence assays based on aptamer/molecularly imprinted polymer/fluorescent probes, electrochemical sensors, and Raman spectroscopy, are thoroughly reviewed and discussed. The focus extends to detecting fipronil and its metabolites in crops, fruits, vegetables, animal-derived foods, water, and bodily fluids. This comprehensive exploration contributes valuable insights into the field, aiming to foster the development and innovation of more sensitive, rapid, and applicable analytical methods.

Generating Monoclonal Antibodies against Buprofezin and Developing Immunoassays for Its Residue Detection in Tea Samples.

The synthesis of a hapten and antigen for the preparation of a monoclonal antibody (mAb) for buprofezin is described. The recognition mechanism of hapten and buprofezin by monoclonal antibodies (mAb-19F2) is described. The effectiveness of the mAb-19F2 immunoassay technique was assessed, and the effective detection of buprofezin in tea samples was achieved through the establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA). The mAb-19F2 subtype was IgG1, with an IC50 of 1.8 ng/mL and a linear range (IC20-IC80) of 0.6-5.4 µg/L, and had a cross-reaction rate of less than 0.18% with 29 other pesticides (neonicotinoids and insect growth regulators). The study identified π-π stacking interactions between hapten and TYR-61 at the mAb-19F2 site and alkyl/phosphate interactions with TRP-105 and ARG-103. The ic-ELISA had an IC50 of 12.9 ng/mL in green tea and 5.65 ng/mL in black tea, with a recovery rate of 92.4%-101.0% and RSD of 2.1%-4.8%. The GICA had a limit of detection (LOD) was 500 ng/mL, with the complete disappearance of the test lines visible to the naked eye. The limit of quantitation (LOQ, IC20) was determined to be 16.8 ng/mL. Additionally, the developed GICA showed no cross-reactivity with neonicotinoid pesticides. The recovery rate of tea spiked recovered samples was 83.6%-92.2%, with an RSD of 5.3%-12.6%, and the results were consistent with the LC/MS method. This study is important for the real-time detection of buprofezin residues to ensure food safety and human health.

Application of Immunoassay Technology in Food Inspection.

Food safety is as important as ever, and the safeguards implemented to inspect and reduce pesticides, veterinary drugs, toxins, pathogens, illegal additives, and other deleterious contaminants in our food supply has helped improve human health and increase the length and quality of our lives [...].

Production of a Monoclonal Antibody for the Detection of Forchlorfenuron: Application in an Indirect Enzyme-Linked Immunosorbent Assay and Immunochromatographic Strip.

In this study, a monoclonal antibody (mAb) specific to forchlorfenuron (CPPU) with high sensitivity and specificity was produced and designated (9G9). To detect CPPU in cucumber samples, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold nanobead immunochromatographic test strip (CGN-ICTS) were established using 9G9. The half-maximal inhibitory concentration (IC50) and the LOD for the developed ic-ELISA were determined to be 0.19 ng/mL and 0.04 ng/mL in the sample dilution buffer, respectively. The results indicate that the sensitivity of the antibodies prepared in this study (9G9 mAb) was higher than those reported in the previous literature. On the other hand, in order to achieve rapid and accurate detection of CPPU, CGN-ICTS is indispensable. The IC50 and the LOD for the CGN-ICTS were determined to be 27 ng/mL and 6.1 ng/mL. The average recoveries of the CGN-ICTS ranged from 68 to 82%. The CGN-ICTS and ic-ELISA quantitative results were all confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with 84-92% recoveries, which indicated the methods developed herein are appropriate for detecting CPPU in cucumber. The CGN-ICTS method is capable of both qualitative and semiquantitative analysis of CPPU, which makes it a suitable alternative complex instrument method for on-site detection of CPPU in cucumber samples since it does not require specialized equipment.

Competitive fluorescent immunosensor based on catalytic hairpin self-assembly for multiresidue detection of organophosphate pesticides in agricultural products.

Simple and rapid multiresidue trace detection of organophosphate pesticides (OPs) is extremely important for various reasons, including food safety, environmental monitoring, and national health. Here, a catalytic hairpin self-assembly (CHA)-based competitive fluorescent immunosensor was developed to detect OPs in agricultural products, involving enabled dual signal amplification followed by a CHA reaction. The developed method could detect 0.01-50 ng/mL triazophos, parathion, and chlorpyrifos, with limits of detection (LODs) of 0.012, 0.0057, and 0.0074 ng/mL, respectively. The spiked recoveries of samples measured using this assay ranged from 82.8 % to 110.6 %, with CV values ranging between 5.5 % and 18.5 %. This finding suggests that the CHA-based competitive fluorescent immunosensor is a reliable and accurate method for detecting OPs in agricultural products. The results correlated well with those obtained from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, indicating that the CHA-based biosensor is able to accurately detect OPs and can be used as a reliable alternative to the LC-MS/MS method. Additionally, the CHA-based biosensor is simpler and faster than LC-MS/MS, which makes it a more practical and cost-effective option for the detection of OPs. In summary, the CHA-based competitive fluorescent immunosensor can be considered a promising approach for trace analysis and multiresidue determination of pesticides, which can open up new horizons in the fields of food safety, environmental monitoring, and national health.

High-throughput analysis of polyethoxylated tallow amine homologs in citrus using a modified QuEChERS-HILIC-MS method.

A new method is described based on ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC) with electrospray mass spectrometry detection for comprehensive quantitative analysis of 66 polyethoxylated tallow amine (POE-tallowamine) homologs in citrus. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing an acetonitrile-aqueous solution containing a 10 mM ammonium formate gradient on a hydrophilic interaction chromatography (HILIC) column with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. The quantitative accuracy and precision of the method were improved by the use of matrix-matched calibration standards. At spiked levels of (50 + 250) µg/kg, (200 + 1000) µg/kg, and (500 + 2500) µg/kg POE-5 and POE-15 (1:5), the average recoveries of the POE-tallowamine homologs ranged from 71.9 to 112%, with RSDs < 16.6%. The limits of detection (LODs) and limits of quantification (LOQs) for POE-tallowamine homologs were 0.01-2.57 and 0.03-8.58 µg/kg, respectively. The method was successfully applied to determine POE-tallowamine in citrus samples from typical Chinese regions in 2021. POE-tallowamine was detected in all 54 samples, and the highest concentration (143 µg/kg) of POE-tallowamine was found in Jelly orange from Zhejiang Province, which might indicate a higher usage and demand of glyphosate herbicides in Zhejiang.

Different dissipation potential and dietary risk assessment of tristyrylphenol ethoxylates in cowpea ecosystem in China.

Tristyrylphenol ethoxylates (TSPEOn) are widely used as inert ingredients in pesticide formulations in the world. However, the information on the dissipation behavior of different homologs TSPEOn in agro-products is lacking. To investigate the dissipation behavior of TSPEOn, a cowpea field experiment treated with TSPEOn at different doses was carried out in Guangdong province, China. Different 24 TSPEO homologs were all detected in cowpea from the field terminal residue experiments, and the total concentrations of TSPEO homologs in cowpea were 40.0-1,374 µg/kg. The dissipation half-lives of 24 TSPEO homologs in soil were 1.51-2.35 times longer than those in cowpea. The long-chain homologs TSPEOn were dissipated faster than the short-chain homologs TSPEOn, suggesting a homolog-specific degradation of the TSPEOn in the cowpea ecosystem. The characteristic bimodal profiles of TSPEOn (n = 6-29) differing from that of the commercial TSPEOn were observed in the cowpea terminal residues experiment, indicating that the long-chain TSPEOn would degrade to short-chain TSPEOn in cowpea and soil. The acute and chronic dietary exposure risks of ΣTSPEOn in cowpea are within acceptable margins for human consumption across different ages and genders. But the health risks to children should be noticed in future.

Development of a time-resolved fluorescence microsphere Eu lateral flow test strip based on a molecularly imprinted electrospun nanofiber membrane for determination of fenvalerate in vegetables.

Fenvalerate residues in fruits and vegetables may result in biological immune system disorders. Current sensor detection methods are harsh due to the shortcomings of antibody preparation and preservation conditions. Therefore, developing a recognition material with strong specificity, good stability, and low cost is of practical significance in designing a sensitive, simple, and rapid method. This study used precipitation polymerization to synthesize molecularly imprinted polymers (MIPs). The MIP was prepared into a fiber membrane using the electrostatic spinning method. After that, the fenvalerate hapten-mouse IgG-Eu fluorescent probe was synthesized, and the side flow chromatography strip was constructed to determine fenvalerate in vegetables using the immunocompetition method. The results showed that the adsorption capacity of MIP to fenvalerate was 3.65, and the adsorption capacity on MIPFM (an electrospinning membrane containing the fenvalerate MIPs) was five times that of free MIP. The test strip showed good linearity with R 2 = 0.9761 within the range of 50 µg/L-1,000 µg/L. In conclusion, substituting fenvalerate monoclonal antibodies with a molecularly imprinted electrospinning membrane is ideal for rapid onsite detection of pyrethroids.

Recent Advances in Rapid Detection Techniques for Pesticide Residue: A Review.

As an important chemical pollutant affecting the safety of agricultural products, the on-site and efficient detection of pesticide residues has become a global trend and hotspot in research. These methodologies were developed for simplicity, high sensitivity, and multiresidue detection. This review introduces the currently available technologies based on electrochemistry, optical analysis, biotechnology, and some innovative and novel technologies for the rapid detection of pesticide residues, focusing on the characteristics, research status, and application of the most innovative and novel technologies in the past 10 years, and analyzes challenges and future development prospects. The current review could be a good reference for researchers to choose the appropriate research direction in pesticide residue detection.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching.

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

A review on recent advances in the applications of composite Fe3O4 magnetic nanoparticles in the food industry.

Fe3O4 magnetic nanoparticles (MNPs) have attracted tremendous attention due to their superparamagnetic properties, large specific surface area, high biocompatibility, non-toxicity, large-scale production, and recyclability. More importantly, numerous hydroxyl groups (-OH) on the surface of Fe3O4 MNPs can provide coupling sites for various modifiers, forming versatile nanocomposites for applications in the energy, biomedicine, and environmental fields. With the development of science and technology, the potential of nanotechnology in the food industry has also gradually become prominent. However, the application of composite Fe3O4 MNPs in the food industry has not been systematically summarized. Herein, this article reviews composite Fe3O4 MNPs, including their properties, modifications, and physical functions, as well as their applications in the entire food industry from production to processing, storage, and detection. This review lays a solid foundation for promoting food innovation and improving food quality and safety.

Design and Characterization of a Novel Hapten and Preparation of Monoclonal Antibody for Detecting Atrazine.

This study provides the first design and synthetic protocol for preparing highly sensitive and specific atrazine (ATR) monoclonal antibodies (mAbs). In this work, a previously unreported hapten, 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine, was designed and synthesized, which maximally exposed the characteristic amino group ATR to an animal immune system to induce the expected antibody. The molecular weight of the ATR hapten was 259.69 Da, and its purity was 97.8%. The properties of the anti-ATR mAb were systematically characterized. One 9F5 mAb, which can detect ATR, was obtained with an IC50 value (the concentration of analyte that produced 50% inhibition of ATR) of 1.678 µg/L for ATR. The molecular weight for the purified 9F5 mAb was approximately 52 kDa for the heavy chain and 15 kDa for the light chain. The anti-ATR mAb prepared in this study was the IgG1 type. The working range of the standard curve (IC20 (the concentration of analyte that produced 20% inhibition of ATR)-IC80 (the concentration of analyte that produced 80% inhibition of ATR)) was 0.384 to 11.565 µg/L. The prepared anti-ATR mAb had high specificity, sensitivity, and affinity with low cross-reactivity. The prepared anti-ATR mAb could provide the core raw material for establishing an ATR immunoassay.

Enzyme inhibition methods based on Au nanomaterials for rapid detection of organophosphorus pesticides in agricultural and environmental samples: A review.

Background: Organophosphorus pesticides (OPs), as insecticides or acaricides, are widely used in agricultural products to ensure agricultural production. However, widespread use of OPs leads to environmental contamination and significant negative consequences on biodiversity, food security, and water resources. Therefore, developing a sensitive and rapid method to determine OPs residues in different matrices is necessary. Originally, the enzyme inhibition methods are often used as preliminary screens of OPs in crops. Many studies on the characteristic of Au nanomaterials have constantly been emerging in the past decade. Combined with anisotropic Au nanomaterials, enzyme inhibition methods have the advantages of high sensitivity, durability, and high stability. Aim of Review: This review aims to summarize the principles and strategies of gold (Au) nanomaterials in enzyme inhibition methods, including colorimetric (dispersion, particle size of Au nanomaterials) and fluorometric (fluorescence energy transfer, internal filtration effect) detection, and electrochemical sensing system (shape of Au nanomaterials, Au nanomaterials combined with other nanomaterials). The application of enzyme inhibition in agricultural products and research progress was also outlined. Next, this review illustrates the advantages of Au nanomaterial-based enzyme inhibition methods compared with conventional enzyme inhibition methods. The detection limits and linear range of colorimetric and fluorometric detection and electrochemical biosensors have also been provided. At last, key perspectives, trends, gaps, and future research directions are proposed. Key Scientific Concepts of Review: Herein, we introduced the technology of enzyme inhibition method based on Au nanomaterials for onsite and infield rapid detection of organophosphorus pesticide.

Residue, Dissipation Pattern, and Dietary Risk Assessment of Imidacloprid in Chinese Chives.

The demand for Chinese chives is growing as they are also rich in vitamins, fiber, and sulfur nutrients. Chinese chives should be sprayed with imidacloprid to control pests and diseases to safeguard their yield and to meet the demands of East Asian consumers for Chinese chives. Overspraying of imidacloprid can lead to residues in Chinese chives, posing a severe risk to human health. To reduce the harmful effects of imidacloprid residues on humans, we investigated the imidacloprid dissipation pattern and the final residue on Chinese chives using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Good linearity (R 2= 0.9988), accuracy (expressed as recovery % of 78.34-91.17%), precision [expressed as relative SDs (RSDs) of 0.48-6.43%], and sensitivity [a limit of quantification (LOQ) ≤ 8.07 × 104 mg/kg] were achieved. The dissipation dynamics were consistent with the first-order kinetics, with a half-life of 2.92 days. The final residual levels on Chinese chives were 0.00923-0.166 mg/kg, which is lower than the maximum residue limits (MRLs) of 1 mg/kg for imidacloprid on Chinese chives. A risk assessment index of <1 indicates that Chinese chives are safe for consumption.

A competitive immunoassay for detecting triazophos based on fluorescent catalytic hairpin self-assembly.

A rapid detection method is introduced for residual trace levels of triazophos in water and agricultural products using an immunoassay based on catalytic hairpin self-assembly (CHA). The gold nanoparticle (AuNPs) surface was modified with triazophos antibody and sulfhydryl bio-barcode, and an immune competition reaction system was established between triazophos and its ovalbumin-hapten (OVA-hapten). The bio-barcode served as a catalyst to continuously induce the CHA reaction to achieve the dual signal amplification. The method does not rely on the participation of enzymes, and the addition of fluorescent materials in the last step avoids interfering factors, such as a fluorescence burst. The emitted fluorescence was detected at 489/521 nm excitation/emission wavelengths. The detection range of the developed method was 0.01-50 ng/mL for triazophos, and the limit of detection (LOD) was 0.0048 ng/mL. The developed method correlates well with the results obtained by LC-MS/MS, with satisfactory recovery and sensitivity. In sum, the designed method is reliable and provides a new approach to detect pesticide residues rapidly and quantitatively.

A Competitive Assay Based on Dual-Mode Au@Pt-DNA Biosensors for On-Site Sensitive Determination of Carbendazim Fungicide in Agricultural Products.

Carbendazim (CBZ), a systemic, broad-spectrum benzimidazole fungicide, is widely used to control fungal diseases in agricultural products. Its residues might pose risks to human health and the environment. Therefore, it is warranted to establish a rapid and reliable method for its residual quantification. Herein, we proposed a competitive assay that combined aptamer (DNA) specific recognition and bimetallic nanozyme gold@platinum (Au@Pt) catalysis to trace the CBZ residue. The DNA was labeled onto bimetallic nanozyme Au@Pt surface to produce Au@Pt probes (Au@Pt-DNA). The magnetic Fe3O4 was functionalized with a complementary strand of DNA (C-DNA) to form Fe3O4 probes (Fe3O4-C-DNA). Subsequently, the CBZ and the Fe3O4 probes competitively react with Au@Pt probes to form two Au@Pt-DNA biosensors (Au@Pt-ssDNA-CBZ and Au@Pt-dsDNA-Fe3O4). The Au@Pt-ssDNA-CBZ biosensor was designed for qualitative analysis through a naked-eye visualization strategy in the presence of CBZ. Meanwhile, Au@Pt-dsDNA-Fe3O4 biosensor was developed to quantitatively analyze CBZ using a multifunctional microplate reader. A competitive assay based on the dual-mode Au@Pt-DNA biosensors was established for onsite sensitive determination of CBZ. The limit of detection (LOD) and recoveries of the developed assay were 0.038 ng/mg and 71.88-110.11%, with relative standard deviations (RSDs) ranging between 3.15 and 10.91%. The assay demonstrated a good correlation with data acquired from liquid chromatography coupled with mass spectrometry/mass spectrometry analysis. In summary, the proposed competitive assay based on dual-mode Au@Pt-DNA biosensors might have a great potential for onsite sensitive detection of pesticides in agro-products.

pH-Responsive On-Demand Alkaloids Release from Core-Shell ZnO@ZIF-8 Nanosphere for Synergistic Control of Bacterial Wilt Disease.

Developing an effective and safe technology to control severe bacterial diseases in agriculture has attracted significant attention. Here, ZnO nanosphere and ZIF-8 are employed as core and shell, respectively, and then a pH-responsive core-shell nanocarrier (ZnO-Z) was prepared by in situ crystal growth strategy. The bactericide berberine (Ber) was further loaded to form Ber-loaded ZnO-Z (Ber@ZnO-Z) for control of tomato bacterial wilt disease. Results demonstrated that Ber@ZnO-Z could release Ber rapidly in an acidic environment, which corresponded to the pH of the soil where the tomato bacterial wilt disease often outbreak. In vitro experiments showed that the antibacterial activity of Ber@ZnO-Z was about 4.5 times and 1.8 times higher than that of Ber and ZnO-Z, respectively. It was because Ber@ZnO-Z could induce ROS generation, resulting in DNA damage, cytoplasm leakage, and membrane permeability changes so the released Ber without penetrability more easily penetrated the bacteria to achieve an efficient synergistic bactericidal effect with ZnO-Z carriers after combining with DNA. Pot experiments also showed that Ber@ZnO-Z significantly reduced disease severity with a wilt index of 45.8% on day 14 after inoculation, compared to 94.4% for the commercial berberine aqueous solution. More importantly, ZnO-Z carriers did not accumulate in aboveground parts of plants and did not affect plant growth in a short period. This work provides guidance for the effective control of soil-borne bacterial diseases and the development of sustainable agriculture.