General Bioluminescence Resonance Energy Transfer Homogeneous Immunoassay for Small Molecules Based on Quantum Dots

Here, we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and <i>Renilla</i> luciferase (Rluc) as the donor (QD-BRET) for the determination of small molecules. The ratio of the donor–acceptor that could produce energy transfer varied in the presence of different concentrations of free enrofloxacin (ENR), an important small molecule in food safety. The calculated Förster distance (<i>R</i>₀) was 7.86 nm. Under optimized conditions, the half-maximal inhibitory concentration (IC₅₀) for ENR was less than 1 ng/mL and the linear range covered 4 orders of magnitude (0.023 to 25.60 ng/mL). The cross-reactivities (CRs) of seven representative fluoroquinolones (FQs) were similar to the data obtained by an enzyme-linked immunosorbent assay (ELISA). The average intra- and interassay recoveries from spiked milk of were 79.8–118.0%, and the relative standard deviations (RSDs) were less than 10%, meeting the requirement of residue detection, which was a satisfactory result. Furthermore, we compared the influence of different luciferase substrates on the performance of the assay. Considering sensitivity and stability, coelenterazine-h was the most appropriate substrate. The results from this study will enable better-informed decisions on the choice of Rluc substrate for QD-BRET systems. For the future, the QD-BRET immunosensor could easily be extended to other small molecules and thus represents a versatile strategy in food safety, the environment, clinical diagnosis, and other fields

Fluorescence Polarization Immunoassay Based on a New Monoclonal Antibody for the Detection of the Zearalenone Class of Mycotoxins in Maize

To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening the zearalenone class of mycotoxins in maize, two new monoclonal antibodies with uniform affinity to the zearalenone class and four fluorescein-labeled tracers were prepared. After careful selection of appropriate tracer–antibody pairs in terms of sensitivity and specificity, a FPIA that could simultaneously detect the zearalenone class with similar sensitivity was developed. Under optimum conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 1.89, 1.97, 2.43, 1.99, 2.27, and 2.44 μg/L for zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and zearalanone, respectively. The limit of detection of FPIA for the zearalenone class was around 12 μg/kg in maize, and the recoveries ranged from 84.6 to 113.8%, with coefficients of variation below 15.3% in spiked samples. Finally, the FPIA was applied for screening naturally contaminated maize samples, and the results indicated a good correlation with that of high-performance liquid chromatography–tandem mass spectrometry

Generation of <i>Slx2</i> knockout mice.

<p>A) Schematic of the genomic target sites in the <i>Slx2</i> gene. B) Analysis of CRISPR/Cas9 system efficiency in mouse embryonic stem (ES) cells by using T7E1 assay. pX330 ligated with different gRNAs were transfected into V6.5 ES cells. Targeted region was PCR amplified and then digested by T7 Endonuclease I. M, marker. NC, negative control, mouse ES cells transfected with GFP plasmid. C) gRNA-1 and gRNA-3 worked in mouse embryos. Sequencing results of mouse embryos injected with Cas9 mRNA and <i>Slx2</i> gRNA-1 or gRNA-3 were shown. D) Genotyping results of line 1 and line 11 F1 mice. Cas9-mediated indels lead to frame shift of <i>Slx2</i>. Blue color showed the sequence of gRNAs; protospacer adjacent motifs (PAM) were labeled with purple color; red color showed the modified sequences information after targeting. -, nucleic acid base deletion. E) Western blot analysis of SLX2 expression level in testis from line 1 (#1) and line 11 (#11) mice. WT, wild-type.</p

CASA analysis of sperm motility parameters.

<p>CASA analysis of sperm motility parameters.</p