Nanozyme-integrated alcogel colorimetric sensor for rapid and on-site detection of tert-butyl hydroquinone.

Tert-butyl hydroquinone (TBHQ) stand as one of the most widely used antioxidants in food and daily chemical products. Rapid and sensitive monitoring of TBHQ holds considerable importance in safeguarding human health due to its potential risks. In this study, we devised an alcogel-based colorimetric sensor enabling the portable and visual detection of TBHQ. The Ce-UiO-66 nanozyme exhibiting remarkable oxidase-like activity, was synthesized and characterized, facilitating the catalysis of TBHQ oxidation to 2-tert-butyl-1,4-benzoquinone (TBBQ). The ensuing chromogenic reaction between TBBQ and ethylenediamine produced a stable and colored product, serving as a reliable indicator for the rapid and specific detection of TBHQ. Building upon this discovery, a portable and low-cost colorimetric sensor was fashioned by integrating the nanozyme into κ-carrageenan alcogel, thereby enabling on-site TBHQ detection via a smartphone-based sensing platform. The colorimetric sensor exhibited a detection limit of 0.8 µg mL-1, demonstrating robust performance across various matrices such as edible oils, cosmetics, and surface water. Recoveries ranged from 84.9 to 95.5%, with the sensor's accuracy further validated through gas chromatography-mass spectrometry. Our study presents an effective approach to rapid and convenient monitoring of TBHQ, exhibiting good extensibility and practicability.

A miniaturized biosensor for rapid detection of tetracycline based on a graphene field-effect transistor with an aptamer modified gate.

Tetracycline is a broad-spectrum antibiotic for human, poultry and livestock that may cause health damage when enriched in humans. Therefore, it is essential to create a rapid tetracycline assay with high sensitivity, specificity and portability. In this study, a miniaturized tetracycline biosensor based on aptamer-modified graphene field-effect transistor (Apt-SGGT) was fabricated and two detection strategies using transfer characteristic curves and real-time channel current were established for different circumstances. The detection limits of the two strategies were 2.073 pM and 100 pM, respectively. The biosensor also demonstrated outstanding stability, anti-interference and specificity ability. Finally, the biosensor was employed to detect the content of tetracycline in Skim Milk with outstanding recovery rate. We believe that the miniaturized Apt-SGGT biosensor with appropriate detection strategies will provide an ideal portable sensing platform for many important analytes in food with superior selectivity and sensitivity.

Selective detection of Pb2+ ions based on a graphene field-effect transistor gated by DNAzymes in binding mode.

Heavy metal contamination has become a severe threat to dairy products through contaminated feed and the environment water. Among them, Pb(II) is highly toxic to the human body even under minimal exposure. Therefore, establishing a fast and sensitive Pb2+ detection technology is significant for rapid screening of vast number of dairy products. Hererin, we report the development of a sensitive and selective Pb(II) biosensor based on a solution-gated graphene transistor (SGGT) with the gate modified by Pb2+-dependent DNAzyme probes. It has also been explored that the DNAzymes working in simple binding mode integrate better with the SGGT than those working in normal catalytic mode, showing significantly stronger channel current responses and lower detection limit down to 0.39 µg/L (or 1.9 nM). Finally, the biosensor was practicably applied to the detection of lead ions in pure milk samples with a high recovery rate. We believe that this work reveals the best strategy for integrating metal ion dependent DNAzyme probes with SGGT sensing platforms to selectively and sensitively detect many metal ions.

Ce(IV)-coordinated organogel-based assay for on-site monitoring of propyl gallate with turn-on fluorescence signal.

Propyl gallate (PG) is a commonly used synthetic phenolic antioxidant in foodstuffs and industrial products. Due to the potential health risk of PG, rapid and on-site detection in food and environment samples are important to guarantee human health. Herein, we demonstrated rapid monitoring of PG by a fluorescence turn-on strategy based on a specific fluorogenic reaction between PG and polyethyleneimine (PEI). Specifically, Ce4+ with oxidase-mimicking activity oxidized PG to its oxides, which then reacted with PEI through the Michael addition to generate the fluorescent compound. The proposed fluorogenic reaction had good specificity for PG, which could distinguish PG from other phenolic antioxidants and interferences. Furthermore, portable and low-cost organogel test kits were prepared using poly(ethylene glycol) diacrylate for quantitative and on-site detection of PG via a smartphone-based sensing platform. The organogel-based assay detection limit was 1.0 µg mL-1 with recoveries ranging from 80.2% to 106.2% in edible oils and surface water. Suitability of the developed assay was also validated by high-performance liquid chromatography. Our study provides an effective fluorescent approach to rapid, specific, and convenient monitoring of PG, which is useful for diminishing the risk of PG exposure.

Colorimetric detection of 2-tert-butyl-1,4-benzoquinone in edible oils based on a chromogenic reaction with commercial chemicals.

2-tert-butyl-1,4-benzoquinone (TBBQ) is the major oxidative product of tert-butylhydroquinone which is a widely used antioxidant in edible oils. The biotoxicity of TBBQ is a risk to human health, that the rapid and accurate monitoring of TBBQ is needed. Herein, a specific chromogenic reaction between TBBQ and polyethyleneimine (PEI) could generate adducts with maximum absorption at 478 nm. Amine groups of PEI are prone to link with TBBQ through Michael addition to form colored adducts. A colorimetric method for detecting TBBQ in edible oils was developed based on the aforesaid chromogenic reaction. The linear range for TBBQ was from 3.0 to 100.0 µg g-1, having a limit of detection of 1.8 µg g-1. Recoveries results ranged from 88.4 % to 93.1 %, which had a good agreement with that of high-performance liquid chromatography. Our study provides a rapid and simple strategy for the sensitive detection of TBBQ using commercial chemicals.

Synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for amantadine with high sensitivity in chicken.

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Hapten synthesis, monoclonal antibody production and immunoassay development for direct detection of 4-hydroxybenzehydrazide in chicken, the metabolite of nifuroxazide.

Derivatization is usually employed in immunoassay for detection of metabolites of nitrofurans and avoiding derivatization could be preferable to achieve an efficient screening. In the study, we designed four haptens of 4-hydroxybenhydrazide (HBH), the nifuroxazide metabolite. The effect of hapten structures on antibody affinity were evaluated and one monoclonal antibody was produced by using the Hapten C with a linear alkalane spacer arm. After optimization, an enzyme linked-immunosorbent assay (ELISA) was established with an 50% inhibition concentration of 0.25 ng mL-1 for HBH, which could ensure the direct detection of HBH without derivatization. The limit of detection of the ELISA for HBH was 0.12 µg kg-1 with the recoveries of 90.1-96.2% and coefficient of variation (CV) values lower than 9.1%. In conclusion, we produced several high affinity antibodies to HBH with new designed hapten and developed an icELISA for the direct detection of HBH without derivatization in chicken.

Magnetic assisted fluorescence immunoassay for sensitive chloramphenicol detection using carbon dots@CaCO3 nanocomposites.

Analytical methods with high sensitivities and short assay times are urgently required for the screening of "zero tolerance" hazardous substances in food. Herein, we propose a fluorescent immunoassay for the highly sensitive and rapid analysis of chloramphenicol (CAP) based on carbon dots (CDs)-encapsulated CaCO3 nanospheres and magnetic nanoparticles (MNPs). The fluorescent immunoprobes were prepared by coupling the anti-CAP antibodies to carboxymethyl cellulose-functional CDs@CaCO3 nanospheres. Chitosan-modified MNPs with "core-shell" structures were prepared and then conjugated to the CAP hapten, acting as the nano-carrier and interface for the immunoreaction. With the assistance of MNPs, the established fluorescent immunoassay achieved the sensitive detection of CAP in chicken with a limit of detection of 0.03 µg kg-1 and recoveries ranging from 83.7%-105.0%. The analysis results of the fluorescent immunoassay were evaluated by the enzyme-linked immunosorbent assay, having a correlation coefficient of 0.981. Our work provides a rapid, facile, and reliable strategy for the highly sensitive analysis of food contaminants based on "green" fluorescent nanoprobes.

Homogeneous fluorescent immunoassay for the simultaneous detection of chloramphenicol and amantadine via the duplex FRET between carbon dots and WS2 nanosheets.

Herein, we proposed a duplex and homogeneous fluorescent immunoassay for the simultaneous detection of amantadine (AMD) and chloramphenicol (CAP) residue in chicken breast with both high sensitivity and short assay time. The immunoassay was based on the fluorescence resonance energy transfer (FRET) between hapten-labeled carbon dots (CDs) and antibody-modified WS2 nanosheets. To achieve the duplex FRET, polyethyleneimine-functionalized blue and green emissive CDs with separated emission were synthesized via a one-pot hydrothermal method and directly coupled with the haptens of AMD and CAP, serving as the energy donors. The antibodies were modified on the surface of WS2 nanosheets with high quenching efficiency to construct the energy acceptor. The specific immunoreaction could trigger the efficient FRET between the donors and the acceptors, causing the fluorescence quenching of CDs. The developed immunoassay was applied to simultaneously detect AMD and CAP, having the detection limit of 0.10 ng g-1 and 0.06 ng g-1, respectively.

Production of a specific monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in biological samples.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 µg/L, 0.32 µg/L, and 0.55 µg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis. Graphical abstract.

Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs.

Immunoassays with ultra-high sensitivity for the rapid detection of chemical contaminants in food are urgently required. However, conventional enzyme-linked immunosorbent assay (ELISA) usually suffer from the moderate sensitivity. Herein, we aim to improve the sensitivity of conventional ELISA by employing the fluorescent carbon dots (CDs) as the signal probes based on the principle of inner filter effect (IFE). In this strategy, the enzymatically formed products of horseradish peroxidase/alkaline phosphatase efficiently quenched the CDs via the IFE. The absorption signal of the conventional ELISA was converted into the fluorescence signal. The fluorescent immunoassay was successfully developed and used to detect amantadine residues in chicken, achieving a limit of detection of 0.02 ng mL-1. The fluorescent immunoassay is a straightforward, extendable and general strategy and exhibits potential in detecting trace amounts of chemical contaminants in foodstuff.

Design, synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for the rapid detection of ractopamine in pork.

In this study, 10 fluorescein-labeled ractopamine (RAC) derivatives (tracers) were synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of RAC in pork, using previously produced RAC polyclonal antibodies. The effect of the tracer structure on the sensitivity of the FPIA was investigated. The specificity of the FPIA was evaluated with 70 ß-agonists and ß-blockers. The FPIA showed a limit of detection of 0.56 µg kg-1 for RAC in pork, with recoveries ranging from 74.8% to 86.6% in spiked samples. The total analysis time, including sample pretreatment, was less than 1 h. The FPIA was used to screen 150 commercial pork samples for RAC residues and the results were consistent with those of an enzyme-linked immunosorbent assay (ELISA) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results demonstrate that the FPIA developed here is a rapid, accurate, and sensitive screening method for RAC residues in pork.

Generic Hapten Synthesis, Broad-Specificity Monoclonal Antibodies Preparation, and Ultrasensitive ELISA for Five Antibacterial Synergists in Chicken and Milk.

An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immunoassay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using trimethoprim as the starting molecule. These haptens contained carboxyl groups on the meta position of trimethoxybenzene for conjugating to protein, while, the common moiety of ASGs, i.e., diaminopyrimidine, was intentionally and maximally exposed to the immune system in animals in order to induce antibodies with broad specificity against ASGs. Five monoclonal antibodies (mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm, named Hapten A, showed not only uniform broad specificity but also high affinity to all five ASGs. We further determined the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. An indirect competitive ELISA (icELISA)-based 5C4 was established and exhibited IC50 values of 0.067-0.139 µg L-1 with cross-reactivity of 48.2%-418.7% for the five ASGs in buffer under optimal conditions. The calculated limits of detection of the icELISA for chicken and milk were 0.06-0.8 µg kg-1 and 0.05-0.6 µg L-1, respectively. The recoveries in spiked chicken and milk samples were 75.2%-101.4% with a coefficient of variation less than 14.3%. In summary, we have developed, for the first time, a rapid and reliable icELISA for ASGs with significantly improved sensitivity and class selectivity.

Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.

BACKGROUND: Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. METHODS: The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. FINDINGS: Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10(-1) to 10(-3) cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011-14, and 16 (1%) of 1322 samples from inpatients with infection. INTERPRETATION: The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. FUNDING: Ministry of Science and Technology of China, National Natural Science Foundation of China.