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Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.
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Bacillus cereus (B. cereus) is a foodborne pathogen that can produce tripartite enterotoxins, which can cause a variety of diseases after infection. It is critical to rapidly and accurately detect strains with enteropathogenic potential to safeguard human health. In this study, a dual-signal visualized detection platform with fluorescence assay and paper-based lateral flow assay (LFA) based on recombinase polymerase amplification (RPA), CRISPR/Cas12a system, and self-developed CRISPR nucleic acid test strips was constructed for enterotoxigenic B. cereus. The genes that encode two tripartite enterotoxinsânheA, nheB, and nheC for nonhemolytic enterotoxin and hblA, hblC, and hblD for hemolysin BLâwere utilized as detection targets. The platform was capable of detecting six enterotoxin genes at the same genomic DNA level. The limits of detection for each gene were 10-3 ng/µL in fluorescence assay and 10-4 ng/µL in LFA. Furthermore, 101-102 CFU/mL of B. cereus in pure culture was detected. Additionally, a smartphone miniprogram could assist in evaluating the results in LFA. The platform demonstrated good utility by detecting B. cereus in food samples, including milk and rice. The results indicate that our RPA-CRISPR/Cas12a dual-signal visualized detection platform can quickly and easily detect B. cereus with three-component enterotoxin-producing potentials. The whole analytic process took less than 60 min without complex operation or expensive equipment.
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The absence of high-affinity antibodies has hindered the development of satisfactory immunoassays for dichlorvos (DDVP) and trichlorfon (TCP), two highly toxic organophosphorus pesticides. Herein, the de novo synthesis of a novel anti-DDVP hapten was introduced. Subsequently, a specific anti-DDVP monoclonal antibody (Mab) was produced with satisfying affinity to DDVP (IC50: 12.4 ng mL-1). This Mab was highly specific to DDVP, and TCP could readily convert into DDVP under mild alkaline conditions. Leveraging this insight, an indirect competitive ELISA was successfully developed for simultaneous detection of DDVP and TCP. The limit of detection in rice, cabbage and apple for DDVP /TCP was found to be 12.1/14.6 µg kg-1, 7.3/8.8 µg kg-1 and 6.9/8.3 µg kg-1, respectively. This study not only provides an effective strategy for producing a high-quality anti-DDVP Mab but also affords a reliable and cost-effective tool suitable for high-throughput detection of DDVP and TCP in food samples.
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Anticorpos Monoclonais , Diclorvós , Ensaio de Imunoadsorção Enzimática , Contaminação de Alimentos , Haptenos , Oryza , Triclorfon , Haptenos/química , Haptenos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Animais , Contaminação de Alimentos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Diclorvós/análise , Oryza/química , Oryza/imunologia , Triclorfon/análise , Triclorfon/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Malus/química , Brassica/química , Brassica/imunologia , Imunoensaio/métodosRESUMO
Gizzerosine is a biogenic amine produced in fish meal drying process and posted higher mortality due to gizzard erosion in poultry than histamine. However, it is difficult to obtain gizzerosine and achieve sensitive practical detection due to its simple structure. Herein, a monoclonal antibody (mAb) specific to gizzerosine was generated based on the new structural design and a fluorescence immunosensor for sensitive and on-site detection of gizzerosine in feed was first established. Molecular modeling of the three-dimensional (3D) structure and surface electrostatic potential of gizzerosine indicated that the carbonyl group of gizzerosine hapten might affect the important sites of antigen-antibody interactions. The proposed structure was used to obtain the sensitive and specific mAb with IC50 of 3.88 ng/mL in indirect competitive ELISA which was approximately 100-fold lower than that of direct competitive ELISA. Considering the practical application scenarios, a fluorescence immunosensor based on microporous dry method integrated with independent quality control line was established to improve detection stability. Under the optimum conditions, the proposed immunosensor showed a good linear relationship from 1.10 to 19.78 ng/mL and provided a low detection limit of 50 ng/g which was approximately 80-fold lower than the maximum recommended amount (0.4 mg/kg) of gizzerosine in feed. The recoveries of 6 kinds of feed ranged from 83.1 % to 114.3 %, which was in good consistence with that of UHPLC-MS/MS. Overall, this work provides a fast, cost-effective and reliable on-site tool for rapid screening of gizzerosine residues in feed samples.
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Ração Animal , Anticorpos Monoclonais , Técnicas Biossensoriais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Ração Animal/análise , Técnicas Biossensoriais/métodos , Limite de Detecção , Animais , Fluorescência , Imunoensaio/métodos , Modelos MolecularesRESUMO
BACKGROUND: Eugenol compounds (EUGs), which share chemical similarities with eugenol, belong to a group of phenolic compounds primarily found in clove oil. They are highly valued by fish dealers due to their exceptional anesthetic properties, playing a crucial role in reducing disease incidence and mortality during the transportation of live fish. Despite their widespread use, the safety of EUGs remains a contentious topic, raising concerns about the safety of aquatic products. This underscores the need for efficient and sensitive analytical methods for detecting EUGs. RESULTS: Nanomaterial-based ratiometric fluorescence immunoassay has gained increasing attention due to its integration of the immunoassay's excellent specificity and compatibility for high-throughput analysis, coupled with the exceptional sensitivity and anti-interference capabilities of ratiometric fluorescence assays. In this study, we developed a sensitive ratiometric fluorescence immunoassay for screening five EUGs. This method employs a broad-specificity monoclonal antibody (mAb) as a recognition reagent, selective for five EUGs. It leverages the horseradish peroxidase (HRP)-triggered formation of fluorescent 2,3-diaminophenazine (DAP) and the quenching of fluorescent gold clusters (Au NCs) for detection. The assay's detection limits for eugenol, isoeugenol, eugenol methyl eugenol, methyl isoeugenol, and acetyl isoeugenol in tilapia fish and shrimp were found to be 9.8/19.5 µg/kg, 0.11/0.22 µg/kg, 19/36 Tilapia ng/kg, 8/16 ng/kg, and 3.0/6.1 µg/kg, respectively. Furthermore, when testing spiked Tilapia fish and shrimp samples, recoveries ranging from 84.1 to 111.9 %, with the coefficients of variation staying below 7.1 % was achieved. SIGNIFICANCE: This work introduces an easy-to-use, broad-specificity, and highly sensitive method for the screening of five EUGs at a pg/mL level, which not only provides a high-throughput strategy for screening eugenol-type fish anesthetics in aquatic products, but also can serve as a benchmark for developing immunoassays for other small molecular pollutants, rendering potent technological support for guarding food safety and human health.
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Eugenol , Ouro , Nanopartículas Metálicas , Eugenol/análise , Eugenol/análogos & derivados , Eugenol/química , Ouro/química , Nanopartículas Metálicas/química , Animais , Imunoensaio/métodos , Limite de DetecçãoRESUMO
Food-borne biotoxins from microbes, plants, or animals contaminate unclean, spoiled, and rotten foods, posing significant health risks. Neutralizing such toxins is vital for human health, especially after food poisoning. Nanobodies (Nbs), a type of single-domain antibodies derived from the genetic cloning of a variable domain of heavy chain antibodies (VHHs) in camels, offer unique advantages in toxin neutralization. Their small size, high stability, and precise binding enable effective neutralization. The use of Nbs in neutralizing food-borne biotoxins offers numerous benefits, and their genetic malleability allows tailored optimization for diverse toxins. As nanotechnology continues to evolve and improve, Nbs are poised to become increasingly efficient and safer tools for toxin neutralization, playing a pivotal role in safeguarding human health and environmental safety. This review not only highlights the efficacy of these agents in neutralizing toxins but also proposes innovative solutions to address their current challenges. It lays a solid foundation for their further development in this crucial field and propels their commercial application, thereby contributing significantly to advancements in this domain.
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Anticorpos de Domínio Único , Animais , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Humanos , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Anticorpos Neutralizantes/imunologia , Toxinas Biológicas/imunologia , Doenças Transmitidas por Alimentos/prevenção & controle , Doenças Transmitidas por Alimentos/imunologia , Camelus/imunologiaRESUMO
Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10-2 ng µL-1 and 102 CFU mL-1, respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.
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Eméticos , Microbiologia de Alimentos , Recombinases/genética , Bacillus cereus/genética , Sistemas CRISPR-Cas , Sensibilidade e Especificidade , Nucleotidiltransferases/genéticaRESUMO
Trimethoprim (TMP), functioning as a synergistic antibacterial agent, is utilized in diagnosing and treating diseases affecting livestock and poultry. Human consumption of the medication indirectly may lead to its drug accumulation in the body and increase drug resistance due to its prolonged metabolic duration in livestock and poultry, presenting significant health hazards. Most reported immunoassay techniques, such as ELISA and immunochromatographic assay (ICA), find it challenging to achieve the dual advantages of high sensitivity, simplicity of operation, and a wide detection range. Consequently, an open droplet microchannel-based magnetosensor for immunofluorometric assay (OMM-IFA) of trimethoprim was created, featuring a gel imager to provide a signal output derived from the highly specific antibody (Ab) targeting trimethoprim. The method exhibited high sensitivity in chicken and pork samples, with LODs of 0.300 and 0.017 ng/mL, respectively, and a wide linear range, covering trimethoprim's total maximum residue limits (MRLs). Additionally, the spiked recoveries in chicken and pork specimens varied between 81.6% and 107.9%, maintaining an acceptable variation coefficient below 15%, aligning well with the findings from the ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. The developed method achieved a much wider linear range of about 5 orders of magnitude of 10-2-103 levels with grayscale signals as the output signal, which exhibited high sensitivity, excellent applicability and simple operability based on magnetic automation.
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Carne de Porco , Carne Vermelha , Animais , Humanos , Suínos , Trimetoprima , Cromatografia Líquida , Galinhas , Espectrometria de Massas em Tandem/métodos , Aves Domésticas , Fluorimunoensaio , Cromatografia Líquida de Alta Pressão/métodosRESUMO
In this study, three eugenol fragment-containing haptens were synthesized, and a monoclonal antibody (mAb) selective for five commonly-found eugenol compounds (EUGs, i.e., eugenol, isoeugenol, methyl eugenol, methyl isoeugenol, and acetyl isoeugenol) was obtained. Based on this mAb, a broad-spectrum indirect competitive ELISA for high-throughput detection of five EUGs was developed. The detection limits for eugenol, isoeugenol, methyl eugenol, methyl isoeugenol and acetyl isoeugenol in both tilapia and shrimp samples were 25.3/ 50.6 µg/kg, 0.075/0.15 µg/kg, 0.48/0.96 µg/kg, 0.16/0.32 µg/kg, and 18.16/36.32 µg/kg, respectively. The recoveries for five EUGs ranged from 80.4 to 114.0 % with a coefficient of variation less than 11.5 %. Moreover, homology modelling and molecular docking were conducted to elucidate the interactions mechanism of mAb-EUGs. The work provides a promising tool for high-throughput screening of EUGs in aquatic products, which can serve as a benchmark for designing haptens and developing immunoassays for other small molecules.
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Immunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection.
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Gizzerosine is responsible for gizzard erosion and black vomit, owing to excessive gastric acid secretion in poultry. It is a biogenic amine that forms during feed processing. Gizzerosine, a derivative of histamine, is a serious threat to animal feed safety and poultry production because it is more potent after ingestion and more harmful to poultry than histamine. The difficulty of obtaining gizzerosine and the lack of simple, rapid, and sensitive in vitro detection techniques have hindered studies on the effects of gizzerosine on gizzard health and poultry production. In this review, we evaluated the natural formation and the chemical synthesis methods of gizzerosine and introduced seven detection methods and their principles for analyzing gizzerosine. This review summarizes the issues of gizzerosine research and suggests methods for the future development of gizzerosine detection methods.
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Galinhas , Histamina , Animais , Imidazóis/farmacologia , Ração Animal/análiseRESUMO
Multicolor-based visual immunosensor is a promising tool for rapid analysis without the use of bulky instruments. Herein, an anti-fenitrothion nanobody-alkaline phosphatase fusion protein (VHHjd8-ALP) was employed to develop a multicolor visual immunosensor (MVIS) and a ratiometric fluorescence MVIS (RFMVIS, respectively). After one-step competitive immunoassay, the VHHjd8-ALP bound to microplate catalyzed phenyl phosphate disodium salt (ArP) into phenol. Under high alkaline condition (pH 12), the phenol reduced KMnO4 to intermediate (K2MnO4) and further to MnO2 in alkaline condition (pH 12), accompanied by a visible color transition of purple-green-yellow, which can be used for semiquantitative visual analysis or qualitative detection by measuring RGB value. RFMVIS was proposed on the basis of MVIS to further improve sensitivity. The CdTe quantum dot and fluorescein were used as signal probes to develop the fluorescent immunosensor. The CdTe dots with red emission (644 nm) was quenched by oxidation of KMnO4, whereas the fluorescein with green emission (520 nm) remained constant, accompanied by a fluorescent color transition of green-yellow-red. By measuring the ratio of the fluorescence intensity (I644/I520), the ratiometric fluorescence immunosensor was developed for qualitative analysis. The two visual immunosensors were sensitive and simple, and they showed good accuracy and practicability in the recovery test, thus are ideal tools for rapid screening.
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Técnicas Biossensoriais , Compostos de Cádmio , Pontos Quânticos , Permanganato de Potássio , Fenitrotion , Fosfatos , Compostos de Manganês , Telúrio , Imunoensaio , Óxidos , Fluoresceínas , Fenóis , Corantes Fluorescentes , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
Cereulide, the exotoxin of emetic Bacillus cereus, has garnered considerable attention due to its capacity to produce foodborne poisonings and great chemical stability. Herein, a G-quadruplex-hemin DNAzyme-based biosensor was developed to detect cereulide in the homogeneous solution. Due to the special ring structure and high affinity to K+, cereulide can be attracted and intercalated into the G-quadruplex; thus, the properties of the G4 DNAzyme can be altered. The melting temperature (Tm) of the G4 DNAzyme in the presence or absence of cereulide was 58.75 and 50.10 °C, respectively, proving the intercalation of cereulide into the G4 DNAzyme. By using the polychromic fluorescence modality of CdTe quantum dots and o-phenylenediamine to assess the variation in the catalytic activity of the DNAzyme, the intercalation of cereulide had bidirectional effects in G4 DNAzyme-mediated reactions, showing that the fluorescence intensity of CdTe quantum dots displayed a linear relationship with the concentration of cereulide from 0.16 to 40 µg/mL with the limit of detection (LOD) of 0.10 µg/mL, while the fluorescence intensity of DAP exhibited a linear relationship with the concentration of cereulide from 0.02 to 40 µg/mL with the LOD of 0.01 µg/mL. It will be a perspective step of controlling cereulide as a hazardous material in food or the environment.
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Compostos de Cádmio , DNA Catalítico , Pontos Quânticos , TelúrioRESUMO
Nanobody (Nb) has gained significant attention in immunoassays owing to its numerous advantages, particularly its ease of molecular evolution. However, the limited understanding of how high sensitivity and specificity attained for antihapten Nbs hamper the development of high-performance Nbs. Herein, the antiparathion Nb (Nb9) we prepared previously was chosen as the model, and an approach based on X-ray crystallography, molecular docking, and rational site-directed saturation mutation for constructing a rapid and effective platform for nanobody evolution was described. Based on the structural analysis, two mutants, namely Nb-D5 (IC50 = 2.4 ± 0.2 ng/mL) and Nb-D12 (IC50 = 2.7 ± 0.1 ng/mL), were selected out from a six-sites directed saturation mutation library, 3.5-fold and 3.1-fold sensitivity enhancement over Nb9 to parathion, respectively. Besides, Nb-D12 exhibited improved sensitivity for quinalphos, triazophos, and coumaphos (5.4-35.4 ng/mL), indicating its broader detection potential. Overall, our study advances an effective strategy for the future rational evolution of Nbs with desirable performance.
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Anticorpos de Domínio Único , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/química , Simulação de Acoplamento Molecular , Sensibilidade e Especificidade , Imunoensaio , Evolução MolecularRESUMO
Inspired by the molecular crowding effect in biological systems, a novel heterogeneous quadratic amplification molecular circuit (HEQAC) was developed for sensitive bimodal miRNA profiling (HEQAC-BMP) by combining an MNAzyme-based DNA nanomachine with an entropy-driven catalytic hairpin assembly (E-CHA) autocatalytic circuit. Utilizing ferromagnetic nanomaterials as the substrate for DNA nanomachines, a biomimetic heterogeneous interface was established; thus, a localized molecular crowding system was created that can elevate the local reaction concentration and accelerate the molecular recognition process for a significant threshold signal. Simultaneously, the threshold signal undergoes further amplification by E-CHA and is transformed into a chemical signal, enabling a colorimetric-fluorescence bimodal signal readout. The HEQAC-BMP enables miRNA detection from 10 aM to 10 nM with detection limits of 3.7 aM (colorimetry) and 4.8 aM (fluorometry), respectively. Moreover, the design principle and strategy of HEQAC-BMP can be customized to address other critical viruses or diseases with life-threatening and socioeconomic impacts, enhancing healthcare outcomes for individuals.
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A heavy-chain antibody (VHH) library against procymidone (PRM) was constructed via immunizing Bactrian camels. Through careful biopanning, seven nanobodies (Nbs) with different sequences were obtained. The variability in their performance was primarily attributed to the amino acid differences in complementarity-determining region 3 (CDR3), as analyzed by molecular docking. The Nb exhibiting the highest sensitivity, named NbFM5, was biotinylated and conjugated to streptavidin-labeled gold nanoparticles to preserve the epitope's activity and prevent a decrease in sensitivity due to traditional random electrostatic adsorption. Subsequently, a simple and sensitive immunochromatographic assay (ICA) was developed for rapid detection of PRM based on biotinylated Nb (btNb). The developed btNb-ICA showed a cut-off value of 200 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 6.04 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.88 ng/mL. The recoveries in actual samples of crops ranged from 82.2 to 117.3%, aligning well with the results obtained from GC-MS/MS (R2 = 0.995). In summary, the developed btNb-ICA demonstrated high specificity and good accuracy for the rapid detection of PRM residues in vegetables. The total analysis time from preparing the sample to obtaining the result was less than 25 min.
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Ouro , Nanopartículas Metálicas , Animais , Simulação de Acoplamento Molecular , Espectrometria de Massas em Tandem , Produtos Agrícolas , Camelus , ImunoensaioRESUMO
Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.
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Corantes Fluorescentes , Espectrometria de Massas em Tandem , Cromatografia Líquida , Corantes Fluorescentes/química , Imunoensaio/métodos , Antígenos , Tomografia por Emissão de PósitronsRESUMO
Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.
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Anticorpos de Domínio Único , HaptenosRESUMO
The simultaneous determination of carbaryl and its metabolite 1-naphthol is essential for risk assessment of pesticide exposure in agricultural and environmental samples. Herein, several bispecific nanobodies (BsNbs) with different lengths of hydrophilic linkers and junction sites were prepared and characterized for the simultaneous recognition of carbaryl and its metabolite 1-naphthol. It was found that the affinity of BsNbs to the analytes could be regulated by controlling linker length and linking terminal. Additionally, molecular simulation revealed that linker lengths affected the conformation of BsNbs, leading to alteration in sensitivity. The BsNb with G4S linker, named G4S-C-N-VHH, showing good thermal stability and sensitivity was used to develop a bispecific indirect competitive enzyme-linked immunosorbent assay (Bic-ELISA). The assay demonstrated a limit of detection of 0.8 ng/mL for carbaryl and 0.4 ng/mL for 1-naphthol in buffer system. Good recoveries from soil and rice samples were obtained, ranging from 80.0% to 112.7% (carbaryl) and 76.5%-110.8% (1-naphthol), respectively. Taken together, this study firstly provided a BsNb with high sensitivity and efficiency against environmental pesticide and its metabolite, and firstly used molecular dynamics simulation to explore the influence of linker on recognition. The results are valuable for the application of immunoassay with high efficiency in the fields of environment and agriculture.
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Inseticidas , Oryza , Carbaril/análise , Inseticidas/análise , Solo , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
Sensing alkaline phosphatase (ALP) activity with high sensitivity and accuracy is critical for both ALP-related health and food safety supervision and the development of ALP-triggered immunoassay platforms. Herein, an ultrasensitive ratiometric fluorescence (RF) sensing system based on the controllable formation of luminescent polydopamine and efficient quenching of carbon dots was proposed for the ALP activity assay, achieving quantitative detection in the range of 0.01-100 mU/L. Furthermore, this RF sensing system was integrated with an ALP-based ELISA platform to construct an RF-ELISA for benzocaine, a potentially abused anesthetic in edible fish, and ultrasensitive assay at the level of fg/mL was realized. This ratiometric strategy-based platform effectively shields various interferences through the self-calibration effect, thus providing more accurate and reliable quantification results. This study not only offers an efficient method for ultratrace detection of ALP and benzocaine but also proposes a universal platform for ultrasensitive detection of diverse targets in food analysis by replacing the recognition unit.