The widespread use of fluoroquinolone (FQ) antibiotics in veterinary practice, combined with the large diversity of compounds within this class, necessitates the development of rapid, sensitive, and selective methods for monitoring their residues in food products of animal origin. In this study, a multiplex lateral flow test (LFT) was developed for the simultaneous detection of three FQs—difloxacin (DIF), enrofloxacin (ENR), and levofloxacin (LEV). The assay was based on an indirect competitive format employing gold-labeled anti-species antibodies and three monoclonal antibodies with negligible cross-reactivity (<1%). This strategy enabled the use of a single nanolabel conjugate while allowing independent optimization of the concentrations of specific antibodies for competitive binding and of the nanolabel for signal generation. Individual LFTs were first developed separately and then integrated into a single multiplex strip containing three spatially separated test zones, with different zone arrangements comparatively evaluated. The limits of detection for the multiplex LFT were 0.9 ng/mL for DIF, 0.1 ng/mL for ENR, and 0.03 ng/mL for LEV, while the visual cutoffs were 10, 1, and 4 ng/mL, respectively. The assay was completed within 17 min, and its selectivity was confirmed using various combinations of antibiotics. The method was validated using spiked milk, beef, pork, and egg samples following matrix-specific extraction procedures. Recoveries ranged from 82.3% to 110.3%, demonstrating acceptable reproducibility and good agreement with ELISA as the reference method. Overall, the developed LFT represents a simple, rapid, sensitive, and user-friendly tool for on-site multiplex screening of FQs in complex food matrices, thereby contributing to improved food safety monitoring.

Plant-derived foods are a vital component of the human diet, but they pose health risks due to the potential presence of anthraquinones (AQs) and their derivatives, and long-term intake of these substances can lead to health issues. Therefore, there is an urgent need for a method to rapidly and simultaneously detect seven AQs, including AQ, emodin, rhein, chrysophanol, aloe emodin, alizarin, and purpurin. In this study, four haptens were designed based on the common parent nucleus structure of the seven AQs combined with computer-aided simulation analysis. MD was used to analyze the recognition mechanism between AQ mAb-4D7 and the seven AQs, revealing that key amino acid residues (TRP90, ASP99, and TYR31) in the complementarity-determining region of the antibody maintain broad-spectrum recognition of AQs through the combined effects of Pi-Pi stacking, hydrogen bonding, and hydrophobic interactions. Based on AQ mAb-4D7, gold nanoparticles based on lateral flow immunoassay (GNP-LFIA) was developed for detecting the seven AQs in tea, aloe vera, and herbal drinks, with calculated limits of detection of 7.282-34.112 μg/kg, 5.536-27.338 μg/kg, and 5.025-26.319 μg/kg, respectively. In real sample detection, the method exhibited high consistency with the results of liquid chromatography-tandem mass spectrometry confirming the accuracy and stability of GNP-LFIA and providing a reliable technical tool for the on-site rapid screening of multiple AQs in plant-derived foods.

Abstract Accurate quantification of 25-hydroxyvitamin D3 (25(OH)D3), the gold-standard biomarker for vitamin D nutritional assessment, still presents considerable analytical challenges. Although 25(OH)D3 exhibits intrinsic photosensitivity and thermal lability, these properties can be effectively managed via standardized light-shielding and inert-atmosphere operations. Key bottlenecks for its immunoassay lie in empirical-driven antibody screening lacking systematic theoretical guidance and poor discrimination of structurally similar vitamin D metabolites. To address these bottlenecks, we explored the molecular-recognition mechanism of high-affinity monoclonal antibody mAb-1F3 toward its hapten using molecular-docking and binding-pocket analysis. Four key amino-acid residues (PHE 52, PHE 91, TYR 32, ILE 100) within the antibody complementarity-determining region form a cooperative binding network for specific recognition of the steroid nucleus and C25-hydroxyl moiety of 25(OH)D3. π-alkyl interactions from PHE 52, PHE 91 and TYR 32 stabilize the hydrophobic steroid skeleton, while ILE 100 forms a conserved hydrogen bond (2.8 Å) to realize specific recognition of the C25-hydroxyl group. This structurally synergistic binding mechanism endows mAb-1F3 with high affinity (IC50 = 24.8 ng/mL) and favorable metabolite-discriminating capability. Guided by this mechanistic insight, recombinant mAb-1F3 was prepared and integrated into a rapid colloidal-gold lateral-flow immunoassay (LFIA). Standard matrix-specific sample extraction procedures were adopted to mitigate matrix interference induced by the high lipophilicity of 25(OH)D3 in complex samples. The LFIA completes the detection within 5 min and shows satisfactory linear consistency with LC-MS/MS in raw milk, dietary supplement, and human serum matrices. This work establishes a structure-driven strategy for rational antibody characterization and design, provides critical mechanistic support for conventional empirical antibody-screening workflows, and facilitates performance improvement of vitamin D immunoanalytical methods.

As widely applied azole antifungal agents, climbazole (CBZ), clotrimazole (CLZ), and isavuconazole (ISKZ) can persist in environmental media and bioaccumulate in aquatic organisms, thus bringing hidden risks to the ecological environment and aquatic products. Herein, a computer-assisted hapten design was adopted to prepare highly specific monoclonal antibodies, with the half-maximal inhibitory concentrations (IC50) of 1.825, 1.732, and 3.013 ng/mL for CBZ, CLZ, and ISKZ, respectively. Molecular recognition was driven by noncovalent interactions in antibody complementarity-determining regions. Based on this, a quantum dot microspheres-based multiplex lateral flow immunoassay was established for simultaneous detection of the three analytes, with calculated limits of detection in lake water, sludge, and fish of 0.198-1.205, 1.183-3.461, and 3.719-6.291 μg/kg, respectively. Satisfactory agreement with high-performance liquid chromatography-tandem mass spectrometry was validated via both spiked matrix recovery tests and real sample detection. This rapid, simple, and portable method provides a robust and convenient tool for large-scale on-site screening of azole antifungals, supporting efficient multicomponent pollutant monitoring.

Given the widespread use and potential health risks of Napropamide (Nap), establishing a rapid and sensitive detection method for its residues is imperative. Herein, we designed novel haptens H1 and H2 via a computer-assisted strategy. H1, with a long-chain carboxyl extension, generated a high-sensitivity recombinant antibody (rAb) 2A4 (IC50 = 1.64 ng/mL), outperforming hapten H2-derived rAb 3D7 (IC50 = 5.08 ng/mL). Molecular docking (MD) showed that the enhanced hydrogen bond donor capacity of hapten H1 formed a hydrophilic binding pocket, with key interactions (a strong hydrogen bond between SER A-103 and TYR B-96 and pi-pi stacking with TRP B-50) ensuring the high sensitivity of rAb 2A4, validating rational hapten design. To validate the reliability of MD predictions, alanine scanning mutagenesis was further performed in this study, with the experimental results further confirming the critical roles of the key amino acid residues in antibody recognition. Based on rAb 2A4, a dual T-line colloidal gold test strip was developed with a calculated limit of detection of 18.72-36.54 mu g/kg for cabbage, watermelon, soybean, and peanut samples. The quantitative results were consistent with Liquid Chromatography-Tandem Mass methods, demonstrating high sensitivity and user-friendliness, suitable for high-throughput monitoring. Therefore, this study provides a practical tool for rapidly detecting Nap in food.

Computer-aided hapten optimization enabled the generation of a high-affinity anti-BFF monoclonal antibody and the development of a rapid GICA strip for BFF detection in animal-derived products.

2'-Fucosyllactose (2'-FL), a major human milk oligosaccharide, has been demonstrated to support early-life gut maturation and microbial balance. However, the association between 2'-FL, intestinal epithelial development, and gut microbiota remains unclear. In this study, a 3-d-old murine model was established to investigate intestinal epithelial differentiation, variations in gut microbes and metabolism. The results showed that 2'-FL enhanced intestinal barrier function by upregulating the expression of tight junction proteins and increasing goblet cell density in the duodenum, in addition to promoting mucin-2 secretion. Furthermore, 2'-FL stimulated the Lgr5+ intestinal stem cell-Paneth cell axis, resulting in a 4-fold increase in Lgr5 expression and upregulation of the Paneth cell marker lysozyme as well as the progenitor cell marker Musashi, thereby accelerating epithelial regeneration. Microbiota analysis revealed the enrichment of Lactobacillus and Bifidobacterium, accompanied by a significant enhancement in the accumulation of l-arginine and ornithine accumulation, which have been shown to promote intestinal development. These results suggest that 2'-FL promotes intestinal stem cell proliferation and barrier maturation by modulating gut microbial metabolism. These findings provide important insights into the role of 2'-FL in promoting infant gut homeostasis.

Biomarker detection for early disease diagnosis demands materials with high sensitivity and anti-interference capability. However, conventional single-signal nanosensors often fail in complex biological matrices due to autofluorescence and matrix interference. Here, to address these challenges, we report the synthesis of chiral multimodal nanoprobes that integrate circular dichroism, fluorescence and magnetic resonance signals. These probes, constructed using either 'intrinsic chiral nanomaterials' or 'hybrid chiral assemblies', leverage synergistic interactions between chiral frameworks and target analytes to enable orthogonal signal cross-validation. The operation time for their synthesis is usually less than 6 h, includes rapid screening (~1 h) of sensitivity and selectivity through optimization of spectral parameters. This strategy accelerates the development of advanced sensing materials, offering a robust platform for biomarker detection in biomedical applications. The protocol requires familiarity with nanomaterial synthesis, surface functionalization and spectroscopic characterization.

A computationally guided hapten design enables high-performance monoclonal antibodies and a high-sensitivity fluorescent immunochromatographic test strip for rapid, quantitative TEA detection in food.

Arsenic, a potent carcinogen, easily accumulates in vegetable oils via contaminated raw materials and processing procedures, posing severe dietary health risks to humans. Current detection methods either rely on expensive instrumentation with complex operations or suffer from serious matrix interference in high-lipid oil samples. Herein, a paper-based colorimetric test strip was developed for rapid quantitative detection of inorganic arsenic in vegetable oils by modifying the classic Gutzeit method. After systematic optimization of the paper substrate, mercuric bromide concentration and dithiothreitol-assisted sample pretreatment, the strip achieved a limit of detection of 0.05 mg kg-1 over a linear range of 0.05-1 mg kg-1, with the whole analysis process completed within 30 min. Validation with spiked and real vegetable oil samples confirmed high consistency with ICP-MS results, with recoveries of 94.13-109.12%, and subsequent stability tests verified reliable performance after 28 days of storage at 4 °C, 37 °C and 45 °C, offering an efficient, low-cost on-site alternative for arsenic detection in vegetable oils and filling the gap in rapid grassroots screening.

Herein, we report D-/L-penicillamine-mediated magnesium oxide-doped cobalt oxide nanoparticles that exhibit strong mirror-image circular dichroism signals ( ~ 102 mdeg at ~800 nm) and function as effective near-infrared-driven photocatalysts for the enantioselective hydroxylation of tyrosine to dihydroxyphenylalanine. The D-nanoparticles demonstrated pronounced enantioselectivity, consuming 92.48% of L-tyrosine versus only 53.85% of D-tyrosine. Magnesium incorporation proves essential, increasing catalytic activity by 22.78 percentage points relative to undoped cobalt oxide nanoparticles. Mechanistically, Mg incorporation modulates the electronic structure to induce surface oxygen vacancies, which act as electron bridges to facilitate oxygen activation, significantly lowering the reaction barrier. Molecular dynamics simulations further unveil an inverse affinity-activity relationship, where heterochiral pairs enable efficient catalytic turnover through weaker, transient single hydrogen bonds, whereas high-affinity homochiral pairs are sequestered in non-productive states via rigid dual hydrogen bonds. This work establishes a distinct paradigm for rational catalyst design, balancing binding affinity with catalytic mobility.

Cyclic organochlorine chemicals (COCs), including dieldrin, endrin, endosulfan, aldrin, heptachlor, chlordane, and toxaphene, persist globally as carcinogenic and neurotoxic persistent organic pollutants, posing bioaccumulation risks. In this study, we designed two haptens based on the common hexachlorocyclopentadiene and norbornane structural scaffolds shared by these seven COCs. The design rationale was evaluated using computational chemistry assistance and validated through animal immunization, followed by immunological analysis data. Based on the predicted hapten H1, a broad-spectrum monoclonal antibody (2A11) was prepared with a sensitivity as low as 14.91 ng/mL. Through the molecular recognition mechanism, the key amino acid residues, HIS-31 and TYR-33, responsible for the broad-spectrum binding and sensitivity to COCs were elucidated. Subsequently, a rapid and broad-spectrum colloidal gold immunochromatographic assay (GICA) was developed. The visual detection limits for the seven COCs were determined to be 10-100 ng/mL in water and 50-500 ng/g in both fish and soil, respectively. Furthermore, results from the analysis of unknown samples showed a good agreement between GICA and gas chromatography-tandem mass spectrometry. The computer-aided chemistry-based hapten prediction strategy effectively guided the preparation of antibodies for broad-spectrum recognition of COCs, enabling their rapid screening and detection.

Mepiquat chloride (MQ) residues threaten human health and ecological safety, requiring rapid and sensitive monitoring. In this study, five haptens were designed for poorly immunogenic MQ, and MQ-H4/MQ-H5 was identified as the optimal pair via computer-aided analysis and serum validation, generating MQ mAb-5H2 with high affinity and specificity. Based on this mAb, a gold nanoparticle-based lateral-flow immunochromatographic assay was developed for MQ on-site screening, with the calculated limits of detection of 0.034, 0.049, and 0.016 mg/kg in potato, wheat, and soil, respectively. The reliability and accuracy of the method were verified through spiked recovery experiments, showing recoveries from 95.2 to 105.7% and a coefficient of variation of 1.6-7.1%, consistent with the results of high-performance liquid chromatography-mass spectrometry, providing a practical and reliable tool for MQ monitoring and a hapten-driven strategy for high-performance antibody generation and sensitive immunoassays targeting other quaternary ammonium pesticides.

Fluopyram (FPR), a widely used succinate dehydrogenase inhibitor fungicide, has raised potential concerns regarding residue risks, highlighting the importance of developing rapid on-site detection methods. In this study, a novel and unique hapten was designed through computer-assisted simulation, leading to the production of a highly sensitive and specific antibody with a half-maximal inhibitory concentration value of 0.09 ng mL-1. Based on this antibody, a colloidal gold immunochromatographic assay strip method applicable to potato and soybean samples was successfully developed, with a visual limit of detection of 10 μg kg-1. In the analysis of real samples, potato sample No. 9 and soybean samples No. 1 and No. 5 were identified as positive samples. These results corresponded closely to those obtained using the instrument detection method, thereby confirming the reliability of the strips.

Salicylic acid (SA), the primary active metabolite of aspirin, is widely used in clinical practice for its antipyretic and analgesic effects. However, excessive use can cause poisoning, increase the body's metabolic burden and potentially become life-threatening. To overcome the limitations of current detection methods -such as complex and time-consuming procedures, reliance on sophisticated instruments, and the need for trained operators -this study developed a rapid, on-site alternative. A highly specific monoclonal antibody against SA was produced using 5-aminosalicylic acid as a hapten, exhibiting high affinity with an IC5n of 198.59 ng/mL. Based on this antibody, a lateral flow immunoassay strip was established, offering a visual detection limit of 50 ng/mL and a cut-off value of 1000 ng/mL for semi-quantitative analysis. Meanwhile, the results obtained from SA determination using strips showed good consistency with indirect competitive enzyme-linked immunosorbent assay, with recovery rates ranging from 97.28% to 105.81%. This strip provides a simple, efficient tool for rapid screening of SA poisoning at point-of-care settings, including emergency departments and primary healthcare facilities, demonstrating strong potential for practical use.

To address the residue risk of pencycuron in vegetable matrices, this study rationally designed and synthesized a hapten with high structural similarity via computer-aided molecular design. Subsequently, a monoclonal antibody (mAb) 4B6 with high affinity and specificity was successfully generated. Leveraging this antibody, two detection platforms were constructed: an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunoassay (CGIA). The half-maximal inhibitory concentration (IC50) of the developed icELISA was determined to be 1.87 ng/mL (95% CI: 1.83 similar to 1.91 ng/mL). For the CGIA, the visual limit of detection (vLOD) ranged from 2 to 20 ng/g in phosphate-buffered saline and various vegetable matrices. Spiked recovery tests on real samples yielded recoveries between 92.32% and 114.86%, which were in good agreement with results obtained from LC-MS/MS. This study provides a reliable dual-mode immunoanalytical solution for both precise laboratory quantification and rapid on-site screening of pencycuron residues, thereby offering technical support for the surveillance of pencycuron contamination in vegetable matrices.

Organophosphorus pesticides pose significant risks to the ecosystem and human health due to their environmental persistence and potential accumulation in medicinal herbs. This study aimed to develop rapid immunoassays for detecting four phosphorothioate organophosphorus pesticides isofenphos-methyl (Fen), isocarbophos (Car), isofenphos (Phe), and isofenphos-oxon (Oxon) in complex Codonopsis pilosula and Angelica sinensis matrices. Five novel haptens (H1-H5) were synthesized. Among them, a broad-spectrum hapten (H1) was rationally designed through computer-aided modeling, which optimized its spatial conformation, electrostatic potential, and electronic structure for maximum similarity to those of the target pesticides. This hapten yielded a high-affinity monoclonal antibody (mAb 2B1) with excellent sensitivity (IC50 values of 0.78, 1.53, 6.89, and 8.36 ng/mL for Fen, Car, Phe, and Oxon, respectively) and specificity. Homology modeling and molecular docking revealed the structural basis for the mAb's differential affinity; amino acid GLY-99(A), TYR-36(B), and TRP-106(A) play a central role in and could be a key target for subsequent antibody affinity maturation to broaden its cross-reactivity profile against structural analogs. GLY-99(A) and TYR-36(B) are responsible for polar complementarity and precise positioning, while TRP-106(A) contributes to hydrophobic stabilization and energy gain. Based on mAb 2B1, a high-throughput ic-ELISA and a rapid dual-test-line immunochromatographic strip (AuNP-LFIA) were developed; it enabled on-site, visual detection within 15 min, with detection limits compliant with regulatory standards. The liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the positive samples was consistent with the results of the AuNP-LFIA method, further validating the reliability, accuracy, and practical applicability of the developed assay in this study for monitoring phosphorothioate organophosphorus pesticide residues in environmental and agricultural products.

Residues of organic heterocyclic herbicides in food matrices threaten food safety and human health, driving the need for sensitive, on-site, portable, and cost-effective detection. Immunoassays offer portability, rapidity, and low cost, but face challenges such as poor signal-molecule stability and batch-to-batch variability. This article briefly reviews common herbicides, highlights principles of several immunoassay techniques and performance-enhancing strategies, and discusses challenges and prospects of emerging nanomaterial-based immunoassays for food contaminants. With advances in novel nanomaterials, high-throughput screening, and portable detection systems, immunoassays can overcome limitations in sensitivity, portability, reliability, and affordability. These developments are expected to extend immunoassay applications beyond food safety monitoring to environmental regulation and bio-diagnostics, enabling broader on-site and field-deployable solutions.

Gastric cancer poses a formidable therapeutic challenge due to high heterogeneity and aggressive invasiveness. To address this challenge, we first prepared chiral Bi2Mo3O12 nanoparticles (NPs) that exhibited a strong circular dichroism signal of approximately 160 mdeg at 920 nm wavelength. Notably, intraperitoneal injection of L-Bi2Mo3O12 NPs achieved complete tumor clearance, as evidenced by extended survival, maintained body weight, and reduced tumor burden. Mechanistically, L-Bi2Mo3O12 NPs initially activated PI3K-Akt and NF-κB signaling pathways via the Toll-like receptor 2 (TLR2) in splenic macrophages, thereby increasing interleukin-6 (IL-6) production. Notably, reactive oxygen species (ROS) generated from molybdenum valence change further amplified the NF-κB pathway. Subsequently, IL-6 was transported to the tumor site via the circulation to activate the JAK-STAT signaling pathway in mouse forestomach carcinoma (MFC) cells, leading to the upregulation of C-X-C motif chemokine ligand 16 (CXCL16). This CXCL16 then recruited intestinal-derived group 3 innate lymphoid cells (ILC 3s), which elevated CXCL10 expression. Ultimately, CXCL10 activated T cells and natural killer (NK) cells, thereby mediating tumor elimination. These findings highlight L-Bi2Mo3O12 NPs as a promising candidate for tumor immunotherapy.

Forchlorfenuron (FCF), a widely utilized cytokinin-type plant growth regulator, poses a considerable threat to human health and ecological stability when overapplied. This issue has consequently driven an escalating demand for real-time, on-site monitoring technologies capable of detecting FCF residues in agricultural products and environmental matrices. Herein, computational quantum chemical analyses were conducted to elucidate the 3D structures of FCF and four designed haptens, thereby enabling the systematic evaluation of potential epitope masking sites. On the basis of these analyses, two structurally reliable haptens were screened out, which further facilitated the successful development of a highly matrix-tolerant and specific anti-FCF monoclonal antibody (mAb). Based on mAb, the AuNP-labeled double-T immunochromatographic assay was firstly developed to detect FCF in orange, grape, and soil samples. The limits of detection (LOD) were 2.640 µg kg-1, 1.069 µg kg-1, and 3.628 µg kg-1, respectively, with linear ranges of 3.55-296.25 µg kg-1, 1.44-231.37 µg kg-1, and 5.28-438.03 µg kg-1, respectively. Method validation confirmed that the obtained results were in good agreement with those derived from LC-MS/MS, thus verifying that the established double-T immunochromatographic assay is well suited for the rapid, quantitative, and on-site monitoring of FCF residues in food and environmental samples.