Broad-spectrum detection of benzimidazoles with lateral flow immunoassay: A computational chemistry-assisted hapten design strategy and explore of molecular recognition mechanism.

Benzimidazoles (BMZs) are a class of veterinary drugs with a benzimidazole ring, the abuse of which poses a serious threat to ecological balance and human health. Consequently, the development of broad-spectrum antibodies and rapid assays are crucial for detecting BMZs in food samples. Herein, we scientifically designed three hapten structures, predicted the availability of the hapten with computational chemistry, and subsequently verified the broad-spectrum with immunological experiments. A broad-spectrum monoclonal antibody (6F10) was prepared based on the predicted hapten-2. Molecular recognition studies illustrated intricate interactions between mAb 6F10 binding to BMZs attributed to halogen bonds and π-π/π-alkyl interactions, revealing key amino acid sites and demonstrating the reliability of the hapten prediction strategies. Finally, a broad-spectrum, rapid, and sensitive lateral flow immunoassay based on aggregation-induced emission microspheres with high fluorescence intensity was established. The LOD values of the proposed method for eight kinds of BMZs were 0.027, 0.032, 0.058, 0.091, 0.087, 0.246, 0.369, and 0.311 ng mL-1, respectively. In this work, a hapten prediction strategy based on a computational chemistry method effectively guided the preparation of antibodies for broad-spectrum recognition of BMZs, and the molecular recognition studies verified the interaction of mAb 6F10 with BMZs, enabling broad-spectrum and sensitive detection of BMZs in milk.

Enantiomeric separation of flavanone on Chiralpak® IA column and determination of the chiral mechanism.

Thirteen flavanone racemates were successfully separated using a Chiralpak® IA column and isopropanol-hexane (50:50, v/v). The mobile phase flow rate and detection wavelength were 0.5 mL/min and 254 nm. The retention times values ranged from 5.50 and 56.45 min. The values of the retention, separation, and resolution factors ranged from 0.63 to 21.67, 1.12 to 2.45, and 0.13 to 11.94. The docking binding energies ranged from -6.2 to -8.2 kcal/mol, showing enthalpy-determined host-guest complex formation. The molecular docking results and the experimental data were agreed well. The results showed that S-enantiomers had stronger bindings with chiral selectors compared to R-enantiomers. Consequently, the R-enantiomers eluted first followed by S-enantiomers. The reported method is highly useful to determine the enantiomeric composition of the reported flavanone in any sample.

Preparation of Chiral Stationary Phase Based on 1,1'-bi-2-Naphthol for Chiral Enantiomer Separation.

Two enantiomeric novel chiral stationary phases (CSPs) R-3-Amide-BINOL CSP (CSP-1) and S-3-Amide-BINOL CSP (CSP-2) were prepared using (R/S)-1,1'-bi-2-naphthol (BINOL) derivatives as chiral selectors. The structure of CSPs was characterized by nuclear magnetic resonance, scanning electron microscope and elemental analysis. Four chiral solutes were selected under normal phase HPLC conditions to evaluate the chiral separation ability of the two novel CSPs. The effects of mobile phase and acidic additives on enantiomeric separation were investigated. The combination of molecular docking simulation and experimental data has elucidated the crucial role of hydrogen bonds and π-π interactions formed between the analyte and CSP in chiral recognition, and different configurations of CSP can cause enantiomeric elution sequence reversal, indicating that the configuration of chiral selectors in CSP has a significant impact on chiral recognition ability.

Rapid and convenient screening method based on single-chain variable fragments for the detection of restricted monensin in chicken muscle.

A colloidal gold immunochromatographic assay (CGIA) based on single-chain variable fragments (scFvs) has been successfully developed for the detection of monensin (MON). Colloidal gold probes were conjugated to anti-MON scFvs through electrostatic interaction, with the conjugated objects serving as the visual signals. The detection lines were formed by capturing the antibody with MON-OVA. This assay offers a rapid detection time of 15 min, a wide linear range from 2.19 to 10.76 ng mL-1, and boasts high accuracy, precision, and an absence of cross-reactivity. By homology modeling and molecular docking, we predicted the interaction patterns between the scFv and monensin, and the amino acid residues involved in the recognition of MON by the antibody were analyzed. These key amino acid sites are presumed integral to ligand recognition per current interaction models. This hypothesis was confirmed by computer-aided alanine scanning mutation, MM/P(G)BSA molecular dynamics simulation, and in vitro binding experiments. In this study, we successfully developed the scFvs-based CGIA system for rapid and easy quantification of monensin, providing a simple, efficient routine detection of chicken muscle samples.

Molecular mechanism of contactin 2 homophilic interaction.

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

Study of the Enantioselectivity and Recognition Mechanism of Allyl-ß-CD Modified Organic Polymer Monolithic Capillary Column.

Allyl-ß-CD was synthesized and used as the chiral functional monomer to prepare chiral organic polymer monolithic columns in capillary HPLC. First, the enantioselectivity of the prepared allyl-ß-CD modified organic polymer monolithic capillary columns was investigated. Then, the influences of enantioseparation conditions of chiral drugs were further explored. Finally, the recognition mechanism was studied by molecular docking with AutoDock. Complete enantioseparations of four chiral drugs as well as partial enantioseparations of eight chiral drugs have been achieved. Results showed that the RSD values for run-to-run, day-to-day, and column-to-column variations ranged from 1.2% to 4.6%, 1.4% to 4.7%, and 2.0% to 6.1%, respectively. The enantioselectivity factor rather than resolution is correlated with the binding free energy difference between enantiomers with allyl-ß-CD. Furthermore, the abundant ether bonds, hydroxyl groups, and hydrophobic cavities in cyclodextrin are responsible for the enantioseparation ability of the chiral monolithic capillary columns.

General hapten skeleton motivated duplex-immunoassay for emergent bisoxatin adulterants in slimming foods.

Adulterating hazardous bisoxatin (BSO) and bisoxatin acetate (BSOA) in slimming foods poses a threat to public health. A rapid synchronous detection method is urgently needed. Herein, the precise design of four novel haptens based on the general skeleton of BSO and BSOA was driven by computer-chemical visualization strategy, which was used to raise monoclonal antibody (mAb) toward both target compounds. The generated mAb 1F1 recognized BSO and BSOA with maximal half-inhibitory concentration (IC50) of 0.26 and 16.85 ng/mL, respectively. The molecular mechanism governing the duplex-recognition of mAb was elucidated by homology modeling and molecular docking. Finally, an immunochromatography (ICA) was developed for identifying BSO and BSOA, demonstrating a detection capability for screening (CCß) estimated to be 10-500 ng/g in candy tablets, jellies, and oral liquids. This study provides a robust approach for determining adulteration in food and offers insights into hapten design to improve antibody recognition spectrum.

A Bispecific Chimeric Aptamer Design Platform Based on c-MET Aptamer with a Replaceable Redundant Region.

Molecular engineering enables the creation of aptamers with novel functions, but the prerequisite is a deep understanding of their structure and recognition mechanism. The cellular-mesenchymal epithelial transition factor (c-MET) is garnering significant attention due to the critical role of the c-MET/HGF signaling pathway in tumor development and invasion. This study reports a strategy for constructing novel chimeric aptamers that bind to both c-MET and other specific proteins. c-MET was identified to be the molecular target of a DNA aptamer, HF3-58, selected through cell-SELEX. The binding structure and mechanism of HF3-58 with c-MET were systematically studied, revealing the scaffold, recognition, and redundancy regions. Through molecular engineering design, the redundancy region was replaced with other aptamers possessing stem-loop structures, yielding novel chimeric aptamers with bispecificity for binding to c-MET and specific proteins. A chimeric bispecific aptamer HF-3b showed the ability to mediate the adhesion of T-cells to tumor cells, suggesting the prospective utility in tumor immunotherapy. These findings suggest that aptamer HF3-58 can serve as a molecular engineering platform for the development of diverse multifunctional ligands targeting c-MET. Moreover, comprehensive understanding of the binding mechanisms of aptamers will provide guidance for the design of functional aptamers, significantly expanding their potential applications.

Epitope-Shared Hapten Enhancing Antibody Polyreactivity for Simultaneous Immunochromatography of Oxyphenisatin Adulterants.

Given the significant threat posed by oxyphenisatin adulterants (OPHs) in weight-loss foods, simultaneous analysis of the OPHs is necessary. Herein, four novel haptens based on the general epitope shared among the OPHs were raised by computer-aided chemical modeling prediction, with the expectation of eliciting antibody responses targeting three of the OPHs. One obtained monoclonal antibody (mAb) showed maximal half-inhibitory concentration (IC50) of 0.40-12.11 ng/mL for OPHs. The key interaction forces responsible for the corecognition of the OPHs were revealed by the intrinsic molecular mechanism. The developed immunochromatography (ICA) indicated a detection capability for screening (CCß) for OPHs estimated to be 5-600 ng/g in jelly, candy tablets, and oral liquid. Furthermore, the analysis of 15 real samples by our method showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our research not only presented a rapid approach for identifying OPHs adulteration but also proposed an effective hapten prediction strategy to enhance antibody polyreactivity.

Molecularly imprinted beads based on modified cellulose hydrogel:A novel solid phase extraction filler for specific adsorption of camptothecin.

Traditional solid phase extraction (SPE) suffers from a lack of specific adsorption. To overcome this problem, a combination of adsorption method and molecular imprinting technology by polydopamine modification was proposed to realize specific recognition of target compounds in SPE, which is of great significance to improve the separation efficiency of SPE. Cellulose hydrogel beads were prepared by dual cross-linking curing method and modified with polydopamine to make them hydrophilic and biocompatible. Subsequently, cellulose hydrogel-based molecularly imprinted beads (MIBs) were synthesized by surface molecular imprinting technology and used as novel column fillers in SPE to achieve efficient adsorption (34.16 mg·g-1) with specific selectivity towards camptothecin (CPT) in 120 min. The simulation and NMR analysis revealed that recognition mechanism of MIBs involved hydrogen bond interactions and Van der Waals effect. The MIBs were successful used in separating CPT from Camptotheca acuminata fruits, exhibiting impressive adsorption capacity (1.19 mg·g-1) and efficient recovery of CPT (81.54 %). Thus, an environmentally friendly column filler for SPE was developed, offering a promising avenue for utilizing cellulose-based materials in the selective separation of natural products.

Coupled physicochemical dual-crosslinking Ti3C2Tx composite aerogels for the selective adsorption of gallium ions in acid fly ash leaching.

Herein, in order to extract Ga3+ from acid fly ash leaching, we propose a functionalized Ti3C2Tx-based MXene composite aerogel adsorbent for Ga3+ adsorption. The prepared physicochemical dual-crosslinking network aerogel MPHG-40 possesses good Ga3+ adsorption performance (132.52 mg g-1) at the pH of 3 and Ga3+ initial concentration of 50 mg L-1 within 6 h. After five adsorption-desorption cycles, the material shows good mass retention and a 95.65 % retention of its initial adsorption capacity, compared to most reported adsorbents. The optimized adsorbent realized good selective adsorption of Ga3+ against Cu2+, Zn2+, Fe3+, and Al3+ in a simulated acid fly ash leaching with the selective coefficient of 8.63, 96.10, 4.49, and 28.30, respectively. The adsorption may comply with a combined mechanism of physical adsorption, electrostatic interactions, ion-exchange mechanism, and ligand chelation, dominated by chemical adsorption, as identified by theoretical calculations based on density functional theory and experimental data. The three-dimensional solid adsorbent constructed in this study provides a new strategy for selective adsorption of Ga3+, making it possible to be applied to solid waste utilization of fly ash.

Construction of dual-chiral covalent organic frameworks for enantioselective separation.

Developing novel chiral stationary phases (CSPs) with versatility is of great importance in enantiomer separation. This study fabricated a dual-chiral covalent organic framework (PA-CA COF) via successive post-synthetic modifications. The chiral trans-1,2-cyclohexanediamine (CA) and (D)-penicillamine (PA) groups were periodically aligned within nanochannels of the COF, allowing selective recognition of enantiomers through intermolecular interactions. It can be a versatile high-performance liquid chromatography (HPLC) CSP for separating a wide range of enantiomers, including chiral pharmaceutical intermediates and chiral drugs. With separation performance comparable to commercial chiral columns and even greater versatility, the PA-CA COF@SiO2 column held promise for practical applications. Chiral separation results combined with molecular simulation indicated that the mixed mode of PA and CA resulted in the broad separation capability of PA-CA COF. The introduction of the dual-chiral COFs concept opens up a new avenue for chiral recognition and separation, holding great potential for practical enantiomer separation.

Update on chiral recognition mechanisms in separation science.

The stereospecific analysis of chiral molecules is an important issue in many scientific fields. In separation sciences, this is achieved via the formation of transient diastereomeric complexes between a chiral selector and the selectand enantiomers driven by molecular interactions including electrostatic, ion-dipole, dipole-dipole, van der Waals or π-π interactions as well as hydrogen or halogen bonds depending on the nature of selector and selectand. Nuclear magnetic resonance spectroscopy and molecular modeling methods are currently the most frequently applied techniques to understand the selector-selectand interactions at a molecular level and to draw conclusions on the chiral separation mechanism. The present short review summarizes some of the recent achievements for the understanding of the chiral recognition of the most important chiral selectors combining separation techniques with molecular modeling and/or spectroscopic techniques dating between 2020 and early 2024. The selectors include polysaccharide derivatives, cyclodextrins, macrocyclic glycopeptides, proteins, donor-acceptor type selectors, ion-exchangers, crown ethers, and molecular micelles. The application of chiral ionic liquids and chiral deep eutectic solvents, as well as further selectors, are also briefly addressed. A compilation of all published literature on chiral selectors has not been attempted.

Central Chirality and Axial Chirality Recognition of the Enantioselective Antibodies to Herbicide Metolachlor.

Enantioselective antibodies have emerged as efficient tools in the field of chiral chemical detection and separation. However, it is complicated to obtain a highly stereoselective antibody due to the unclear recognition mechanism. In this study, the hapten of metolachlor was synthesized and enantio-separated. The absolute configuration of the four haptens obtained was identified by the computed and experimental electronic circular dichroism comparison. Five polyclonal antibodies against the Rac-metolachlor and its enantiomers were generated by immunization. The cross-activity of all the 5 antibodies with 44 structural analogues, including metolachlor enantiomers, was tested. It demonstrated that antibodies have higher specificity to recognize central chirality than axial chirality. Especially, αRR-MET-Ab exhibited excellent specificity and stereoselectivity. Accordingly, 3D-QSAR models were constructed and revealed that paired stereoisomers exhibited opposite interactions with the antibodies. It is the first time that the antibodies against four stereoisomers were prepared and analyzed, which will be conducive to the rational design of the stereoselective antibodies.

Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development.

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 µg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 µg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Preparation of amino acid chiral ionic liquid and visual chiral recognition of glutamine and phenylalanine enantiomers.

In this paper, the amino acid chiral ionic liquid (AACIL) was prepared with L-phenylalanine and imidazole. It was characterized by CD, FT-IR, 1H NMR, and 13C NMR spectrum. The chiral recognition sensor was constructed with AACIL and Cu(II), which exhibited different chiral visual responses (solubility or color difference) to the enantiomers of glutamine (Gln) and phenylalanine (Phe). The effects of solvent, pH, time, temperature, metal ions, and other amino acids on visual chiral recognition were optimized. The minimum concentrations of Gln and Phe for visual chiral recognition were 0.20 mg/ml and 0.28 mg/ml, respectively. The mechanism of chiral recognition was investigated by FT-IR, TEM, SEM, TG, XPS, and CD. The location of the host-guest inclusion or molecular placement has been conformationally searched based on Gaussian 09 software.

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.

Investigation on improvement of enantioseparation based on clindamycin phosphate by chiral deep eutectic solvents in capillary electrophoresis.

In this work, the potential synergetic effect between deep eutectic solvents and an antibiotic chiral selector (clindamycin phosphate) for enantioseparation was investigated in capillary electrophoresis. We synthesized a series of deep eutectic solvents with choline chloride as hydrogen bond acceptor and three α-hydroxyl acids (l-lactic acid, l-malic acid, and l-tartaric acid) as hydrogen bond donors. Compared to the single clindamycin phosphate separation system, significantly improved separations of model drugs were observed in several synergetic systems. Compared to deep eutectic solvents with a single hydrogen bond donor, deep eutectic solvents with mixed-type hydrogen bond donors were superior. The influences of several key parameters including the type and proportion of organic modifier, clindamycin phosphate concentrations, deep eutectic solvents concentrations, and buffer pH were investigated in detail. The mechanism of the enhanced separations in deep eutectic solvents systems was investigated by means of electroosmotic flow analysis, nuclear magnetic resonance analysis, and molecular modeling. It was the first time that the synergetic systems between deep eutectic solvents and antibiotic chiral selector were established in capillary electrophoresis, and these deep eutectic solvents were demonstrated to have a good synergetic effect with clindamycin phosphate for enantioseparation.

Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay.

Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP-99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP-specific scFv antibodies and also propose a multi-amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.

Establishment of an indirect competitive immunoassay for the detection of dicamba based on a highly specific nanobody.

Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC50 of 0.93 µg/mL and a linear range of 0.11-8.01 µg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.