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.

Novel hapten design, antibody recognition mechanism study, and a highly sensitive immunoassay for diethylstilbestrol in shrimp.

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 µg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. 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.

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.

Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon Nanoparticles for Detecting Three Fusarium Mycotoxins in Maize.

The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 µg/kg for deoxynivalenol, 13 µg/kg for T-2 toxin, and 1 µg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography-tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.