"Four-In-One" Multifunctional Dandelion-Like Gold@platinum Nanoparticles-Driven Multimodal Lateral Flow Immunoassay.

The traditional lateral flow immunoassay (LFIA) with a single signal output mode may encounter challenges such as low sensitivity, poor detection range, and susceptibility to external interferences. These limitations hinder its ability to meet the growing demand for advanced LFIA. To address these issues, the rational development of multifunctional labels for multimodal LFIA emerges as a promising strategy. Herein, this study reports a multimodal LFIA using "four-in-one" multifunctional dandelion-like gold@platinum nanoparticles (MDGP). The inherent properties of MDGP, such as the broad absorption spectrum, porous dandelion-like nanostructure, and bimetallic composition with gold and platinum, endow them with capacities in dual spectral-overlapped fluorescence quenching, optical readout, catalytic activity, and photothermal effect. Benefiting from their multifunctional properties, the MDGP-LFIA enables multimodal outputs including fluorescent, colorimetric, and photothermal signals. This multimodal MDGP-LFIA allows for the detection of acetamiprid at a range of 0.01-50 ng mL-1 , with the lowest qualitative and quantitative detection results of 0.5 and 0.008 ng mL-1 , respectively, significantly better than the traditional gold nanoparticles-based LFIA. The diversity, complementarity, and synergistic effect of integrated output signals in this multimodal MDGP-LFIA improve the flexibility, practicability, and accuracy of detection, holding great promise as a point-of-care testing platform in versatile application scenarios.

Insights into the Binding Profile of Anti-chlorpyrifos Recombinant Antibodies: From Computational Simulation to Immunoassay Validation.

A novel virtual screening strategy was proposed for the profiling and discovery of active variable regions (VRs) that encode hapten-specific recombinant antibodies (rAbs). Chlorpyrifos, a hazardous organophosphorus pesticide, was selected as the target. First, a VR model-14G4 from anti-chlorpyrifos hybridoma was built via homology modeling. Its binding pattern toward seven organophosphorus analogues was assessed through virtual screening by performing molecular docking. Based on energy scoring, visual examination, and molecular interaction analysis, chlorpyrifos-methyl was also inferred as the high-affinity target for model-14G4 and was then confirmed via a non-competitive surface plasmon resonance (SPR) assay. Subsequently, we attempted to discover hapten-specific VRs by creating a collection of VR models for anonymous testing. Chlorpyrifos and model-14G4 were employed as the known hit and active VRs, respectively. After molecular docking, a novel anti-chlorpyrifos VR (model-1) was identified due to its satisfactory energy scoring and a similar binding pattern to the reference model-14G4. Expressed by HEK293(F) mammalian cells, the newly prepared full-length rAb-model-1 and rAb-14G4 exhibited high sensitivities for detecting chlorpyrifos by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), with IC50 of 3.01 ng/mL and 42.82 ng/mL, respectively. They recognized chlorpyrifos-methyl with a cross-reactivity (CR) of 2.5-17.3%. Moreover, the binding properties of rAb-model-1 for recognizing chlorpyrifos and chlorpyrifos-methyl were confirmed via a non-competitive microscale thermophoresis (MST) method. Thus, the experimental results showed good agreement with computational outputs on antibody profiling. Furthermore, the recognition diversity of rAb-model-1 for chlorpyrifos and chlorpyrifos-methyl was studied via molecular dynamics simulation. Overall, the proposed study provides a versatile and economical strategy for antibody characterization and promotes the in vitro production of rAbs for pesticide monitoring.

Computer-aided profiling of a unique broad-specific antibody and its application to an ultrasensitive fluoroimmunoassay for five N-methyl carbamate pesticides.

Pollution of N-methyl carbamate (NMC) pesticides is threatening the non-target organisms' survival. Thus, broad-specific antibodies and class-selective immunoassays are demanding for multiple NMCs determination. In this study, we employed a molecular docking-based virtual screening strategy to fast profile antibody spectrum, based on a designed chemical pool containing 17 compounds. A monoclonal antibody (mAb)-6G against carbofuran was used as the objective. The recombinant full-length IgG was successfully expressed to validate the antibody sequences for homology modeling. After docking, we manually categorized the antibody-chemical binding strength into three groups. Non-competitive surface plasmon resonance (SPR) demonstrated the mAb-6G affinitive binding toward five NMCs (carbofuran, isoprocarb, propoxur, carbaryl and carbosulfan), which were classified into strong and moderate binding categories. Antibody binding properties were confirmed again by ic-ELISA and lateral flow immunochromatographic strip. Subsequently, an ultrasensitive indirect competitive fluoromicrosphere-based immunoassay (ic-FMIA) was established with the IC50 (half-maximal inhibitory concentration) values of 0.08-3.37 ng/mL. This portable assay presented a 30-230-fold improved sensitivity than traditional ic-ELISA and was applied in European surface water analysis. Overall, our work provides an efficient platform integrating in-silico and experimental methodologies to accelerate the characterization of hapten-specific antibody binding properties and the development of high-sensitive immunoassays for multi-pollutants monitoring.

Novel immunochromatographic strip assay based on up-conversion nanoparticles for sensitive detection of imidacloprid in agricultural and environmental samples.

Neonicotinoid insecticides are widely used in agriculture for pest control, but the pesticide residues in environmental and agricultural products were a big threat to the health of non-target organisms. In this study, a new immunochromatographic strip test was established for the rapid detection of imidacloprid residue, a neonicotinoid insecticide, based on up-conversion nanoparticles (UCNPs) coupled with the monoclonal antibody against imidacloprid. Under optimal conditions, the half inhibitory concentration (IC50), detection limit, and the linear range of this strip were 8.37 ng/mL, 0.45 ng/mL, and 0.97-250 ng/mL. The strip test could be completed in 30 min. The average recoveries of imidacloprid spiked in water, Chinese cabbages, cucumber, honey, and tea samples were 70.1~101.8%, with coefficient of variations less than 18.9%. The strip was used to test real samples and verified by UPLC-MS/MS method with the good agreement (R2 was 0.9825), indicating this novel strip immunoassay is accurate and reliable.

A sensitive immunoassay based on fluorescence resonance energy transfer from up-converting nanoparticles and graphene oxide for one-step detection of imidacloprid.

In this study, a fluorescence resonance energy transfer (FRET) immunoassay based on graphene oxide (GO) and up-converting nanoparticles (UCNPs) was established for rapid detection of imidacloprid, a commonly-used insecticide. Under 980 nm near-infrared light excitation, emission of UCNPs at 542 nm can be absorbed by the energy acceptor GO. The carboxyl-functionalized GO and UCNPs were coupled with competitive antigen and antibody against imidacloprid. After optimization, the FRET immunoassay showed a wide detection range of 0.08-50 ng/mL to imidacloprid, with cross-reaction toward other three neonicotinoids including imidaclothiz (74.4%), thiacloprid (36.9%) and clothianidin (31.9%). The average recoveries of spiked water, Chinese cabbage, cucumber, honey and tea samples were 76.8%-101.8%. The accuracy and reliability of the FRET immunoassay were verified by UPLC-MS/MS with a good correlation (R2 = 0.9816). In a summary, this study provides a sensitive and one-step method for monitoring imidacloprid residue in food and environmental samples within 1 h.

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody.

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 µg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

Quantum Dots-Based Immunochromatographic Strip for Rapid and Sensitive Detection of Acetamiprid in Agricultural Products.

In this study, a rapid and sensitive immunochromatographic strip (ICS) assay, based on quantum dots (QDs), was developed for the qualitative and quantitative detection of acetamiprid in agricultural samples. Acetamiprid-ovalbumin conjugates (ACE-OVA) and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane as a test and control line. Two kinds of anti-acetamiprid monoclonal antibodies (mAb) obtained in our lab were characterized by the ELISA and surface plasmon resonance assay. The competitive immunoassay was established using a QDs-mAb conjugate probe. The visual detection limit of acetamiprid for a qualitative threshold was set as 1 ng/mL to the naked eye. In the quantitative test, the fluorescence intensity was measured by a portable strip reader and a standard curve was obtained with a linear range from 0.098 to 25 ng/mL, and the half maximal inhibitory concentration of 1.12 ng/mL. The developed method showed no evident cross-reactivities with other neonicotinoid insecticides except for thiacloprid (36.68%). The accuracy and precision of the developed QDs-ICS were further evaluated. Results showed that the average recoveries ranged from 78.38 to 126.97% in agricultural samples. Moreover, to test blind tea samples, the QDs-ICS showed comparable reliability and a high correlation with ultra-performance liquid chromatography-tandem mass spectrometry. The whole sample detection could be accomplished within 1 h. In brief, our data clearly manifested that QDs-ICS was quite qualified for the rapid and sensitive screening of acetamiprid residues in an agricultural product analysis and paves the way to point-of-care testing for other analytes.

Up-Converting Nanoparticle-Based Immunochromatographic Strip for Multi-Residue Detection of Three Organophosphorus Pesticides in Food.

Organophosphorus (OP) pesticides are widely used to control pests because of their high activity. This study described a rapid and sensitive lateral flow immunochromatographic (LFIC) assay based on up-converting nanoparticles (UCNPs) for multi-residue detection of three OP pesticides. The developed assay integrated novel fluorescent material UCNPs labeled with a broad-specific monoclonal antibody. Based on the competitive platform by immobilized antigen in the test zone, the optimized UCNPs-LFIC assay enabled sensitive detection for parathion, parathion-methyl, and fenitrothion with IC50 of 3.44, 3.98, and 12.49 ng/mL (R 2 ≥ 0.9776) within 40 min. The detectable ability ranged from 0.98 to 250 ng/mL. There was no cross-reactivity with fenthion, phoxim, isocarbophos, chlorpyrifos, or triazophos, even at a high concentration of 500 ng/mL. Matrix interference from various agricultural products was also studied in food sample detection. In the spiked test, recoveries of the three OP pesticides ranged from 67 to 120% and relative standard deviations were below 19.54%. These results indicated that the proposed strip assay can be an alternative screening tool for rapid detection of the three OP pesticides in food samples.

Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor.

In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71 ng mL-1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10-9 M and 4.26 × 10-9 M for parathion, 6.75 × 10-9 M and 4.17 × 10-9 M for methyl-parathion, and 2.44 × 10-8 M and 1.19 × 10-8 M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. Graphical abstract ᅟ.