CRISPR/Cas12a-mediated Enzymatic recombinase amplification for rapid visual quantitative authentication of halal food.

Economically motivated adulteration (EMA) has become a concern in food safety. We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) that integrates Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to detect halal food adulteration. We designed and screened crRNA targeting CLEC, a porcine-specific nuclear single-copy gene, and optimized the reagent concentrations and incubation times for the ERA and Cas12a cleavage steps. CAMERA was highly specific for pork ingredients detection. The DNA concentration and fluorescence signal intensity relationship was linear at DNA concentrations of 20-0.032 ng/µL. CAMERA detected as few as two CLEC copies and quantified samples with porcine DNA content as low as 5% within 25 min. The system could be operated in a miniaturized working mode that requires no technical expertise or professional equipment, making CAMERA a valuable tool in resource-limited areas for the qualitative and quantitative detection of pork ingredients in halal food.

Pb2+ Responsive Cu-In-Zn-S Quantum Dots With Low Cytotoxicity.

Water-soluble Cu-In-Zn-S quantum dots (CIZS QDs) with orange fluorescence have been synthesized with a glutathione (GSH) as stabilizer via facile a one-step hydrothermal method. The optimal reaction conditions of CIZS QDs including temperature, time, pH, and the molar ratios of precursors were studied. TEM results indicate that the aqueous-dispersible CIZS QDs are quasi-spherical, and the average diameters are 3.76 nm with excellent fluorescent stability. Furthermore, the cytotoxicity of CIZS QDs was investigated by the microcalorimetry combining with TEM and the IC 50 was 10.2 µM . CIZS QDs showed a promising perspective in applications such as a fluorescent probe for bioimaging and biolabeling due to the low cytotoxicity and good biocompatibility. Moreover, the CIZS QDs can distinguish Pb2+ ion from other ions, offering great potentials in lead ion determination in drinking water. According to the results of UV, XRD, FL, PL, and ITC methods, the mechanism of CIZS QDs-Pb2+ assay is due to hydrogen bonding or van der Waals forces in the formation of Pb2+ and CIZS QDs.

Development of Anti-Idiotypic Nanobody-Phage Based Immuno-Loop-Mediated Isothermal Amplification Assay for Aflatoxins in Peanuts.

Aflatoxin contamination in agricultural products has posed serious health hazards and brought huge economic loss in the food and feed industries. Monitoring aflatoxins in various foods and feeds has become a crucial means to protect public health. This study aimed to report an immuno-loop-mediated isothermal amplification (iLAMP) assay by using an anti-idiotypic nanobody-phage for on-site and rapid detection of aflatoxin in real samples. The iLAMP method was developed on the basis of a competitive immunoassay and LAMP reaction performed in a simple water bath. This method can provide visualized test results: violet color represents positive samples while sky blue represents negative. The visual detection limits of iLAMP for aflatoxin B1, B2, G1, and G2 in peanut samples were 1.6, 1.6, 3.2, and 16 µg/kg, respectively. The developed assay was verified with high performance liquid chromatography (HPLC) for the analysis of aflatoxins in peanuts, which demonstrated that the iLAMP method can be applied to the detection of aflatoxin in real samples. The novel iLAMP assay eliminates the need for aflatoxin conjugates, the antibody labeling process, and special equipment, and offers an alternative to existing methods with advantages of time-saving, cost-effectiveness, and ease-of-use.

The interaction mechanism between fludarabine and human serum albumin researched by comprehensive spectroscopic methods and molecular docking technique.

Fludarabine (Flu) is widely used to treat B-cell chronic lymphocytic leukemia. HSA is of the essence to human, especially in blood circulation system. The interaction mechanism between Flu and HSA was studied by comprehensive spectroscopic methods and molecular docking technique. UV-vis and FL spectrum results indicated that Flu bond with HSA, and there was a new complex produced at the binding site I in subdomain IIA. Association constants at 298 K were 1.637 × 104 M-1 and 1.552 × 104 M-1 at 310 K, respectively. The negative enthalpy (ΔH) and positive entropy (ΔS) values for the interaction revealed that the binding behavior was driven by hydrophobic forces and hydrogen bonds. The results obtained from UV, RLS spectra, 3D fluorescence and CD spectrum illustrated that Flu could change the secondary structure of HSA. According to molecule docking result, the binding energy of interaction is -11.15 kcal/mol.

Facile one-step synthesis of quaternary AgInZnS quantum dots and their applications for causing bioeffects and detecting Cu2.

Water-soluble AgInZnS quantum dots (AIZS QDs) were synthesized with glutathione (GSH) as a stabilizer by a facile one-step method based on a hydrothermal reaction between the nitrate salts of the corresponding metals and sodium sulfide as a sulfide precursor at 110 °C. The optimal reaction conditions (temperature, time, pH, and the molar ratios of the precursors) were studied. According to the data from TEM, XPS, and XRD, AIZS QDs were characterized with excellent optical properties. The results showed that the aqueous-dispersible AIZS QDs were quasi-spherical and their average diameter was 3.51 nm. Furthermore, the cytotoxicity of AIZS QDs was investigated by microcalorimetry and microscopy techniques (confocal microscopy and TEM). The data revealed that AIZS QDs exhibited low toxicity, biocompatibility, and good water stability, due to which they could be used as a fluorescent probe for bioimaging and labeling. In addition, AIZS QDs could be used as a sensor to detect Cu2+ because the fluorescence of AIZS QDs was quenched by Cu2+.

Cytotoxicity of CdTe quantum dots with different surface coatings against yeast Saccharomyces cerevisiae.

Cadmium (Cd)-based QDs are well studied owing to their excellent optical properties. The applications of Cd-based QDs in biomedical filed, however, is hindered by its inherent toxicity. In this study, to overcome the inherent toxicity of heavy metals, CdTe QDs were encapsulated with different shells (NAC, MPA and GSH) to reduce the leakage of Cd from the core. We studied the cytotoxicity of the three kinds of CdTe QDs on S. cerevisiae by spectroscopic, electrochemical, microscopic methods and microcalorimetric technique. Results showed that toxicity of CdTe QDs increased with the augment of QD concentration. According to the values of IC50 ((GSH-CdTe QDs (15.3 nmol/L) < MPA-CdTe QDs (56.2 nmol/L) < NAC-CdTe QDs (89.8 nmol/L)), the most toxic one is GSH-CdTe QDs, followed by MPA-CdTe QDs, then NAC-CdTe QDs. The coatings have contribution to their toxicity. The three kinds of QDs with the similar shape (sphere) can enter the cell by the clathrin-mediated endocytosis and lead to the different impairments. The mechanism of cytotoxicity is due to the release of Cd2+ leading elevation of intracellular reactive oxygen species (ROS), which damage mitochondria. The clathrin-mediated endocytosis is a significant factor in determining the toxicity of CdTe QDs.

Anti-idiotypic nanobody-phage based real-time immuno-PCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs.

Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (IPCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05 to 122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results (R(2) = 0.991) were obtained. In this context, this study provides a novel opportunity to analyze aflatoxins in agri-products.

Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 µM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.

Development and application of an immunoaffinity column enzyme immunoassay for mycotoxin zearalenone in complicated samples.

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC50 against ZEA of 0.02 µg L(-1). The mAb 2D3 exhibited a high recognition of ZEA (100%) and ß-zearalenol (ß-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and ß-zearalanol (ß-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83-93%) and HPLC (94-108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize.