Development and perspectives of rapid detection technology in food and environment.

Food safety become a hot issue currently with globalization of food trade and food supply chains. Chemical pollution, microbial contamination and adulteration in food have attracted more attention worldwide. Contamination with antibiotics, estrogens and heavy metals in water environment and soil environment have also turn into an enormous threat to food safety. Traditional small-scale, long-term detection technologies have been unable to meet the current needs. In the monitoring process, rapid, convenient, accurate analysis and detection technologies have become the future development trend. We critically synthesizing the current knowledge of various rapid detection technology, and briefly touched upon the problem which still exist in research process. The review showed that the application of novel materials promotes the development of rapid detection technology, high-throughput and portability would be popular study directions in the future. Of course, the ultimate aim of the research is how to industrialization these technologies and apply to the market.

Detection of 4 quinolone antibiotics by chemiluminescence based on a novel Nor-Biotin bifunctional ligand and SA-ALP technology.

A simple and effective direct competitive chemiluminescence immunoassay for the detection of 4 kinds of quinolone antibiotics in milk was established using Nor-Biotin (biotin-modified norfloxacin [NOR]) bifunctional ligand and alkaline phosphatase-conjugated streptavidin signal amplification technology. The polyclonal antibody was obtained after the immunization of New Zealand White rabbits using norfloxacin-derived antigen. "Click chemistry" was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive chemiluminescence assay method was developed and used for sensitive detection of 4 kinds of quinolone drugs (NOR, pefloxacin, ciprofloxacin, and danofloxacin). The IC50 of the 4 kinds of quinolone drugs ranged from 7.35 to 24.27 ng/mL, and the lowest detection limits ranged from 0.05 to 0.16 ng/mL, which were below their maximum residue levels, approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the 4 quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.

State-of-the-art progress of switch fluorescence biosensors based on metal-organic frameworks and nucleic acids.

Metal-organic frameworks (MOFs) have captured substantial attention of an increasing number of scientists working in sensing analysis fields, due to their large surface area, high porosity, and tunable structure. Recently, MOFs as attractive fluorescence quenchers have been extensively investigated. Given their high quenching efficiency toward the fluorescence intensity of dyes-labeled specific biological recognition molecules, such as nucleic acids, MOFs have been widely developed to switch fluorescence biosensors with low background fluorescence signal. These strategies not only lead to specificity, simplicity, and low cost of biosensors, but also possess advantages such as ultrasensitive, rapid, and multiple detection of switch fluorescence methods. At present, researches of the analysis of switch fluorescence biosensors based on MOFs and nucleic acids mainly focus on sensing of different types of in vitro and intracellular analytes, indicating their increasing potential. In this review, we briefly introduce the principle of switch fluorescence biosensor and the mechanism of fluorescence quenching of MOFs, and mainly discuss and summarize the state-of-the-art advances of MOFs and nucleic acids-based switch fluorescence biosensors over the years 2013 to 2020. Most of them have been proposed to the in vitro detection of different types of analytes, showing their wide scope and applicability, such as deoxyribonucleic acid (DNAs), ribonucleic acid (RNAs), proteins, enzymes, antibiotics, and heavy metal ions. Besides, some of them have also been applied to the bioimaging of intracellular analytes, emerging their potential for biomedical applications, for example, cellular adenosine triphosphate (ATP) and subcellular glutathione (GSH). Finally, the remaining challenges in this sensing field and prospects for future research trends are addressed. Graphical abstract.

A low-field nuclear magnetic resonance DNA-hydrogel nanoprobe for bisphenol A determination in drinking water.

A low-field nuclear magnetic resonance (LF-NMR) DNA-hydrogel (LNDH) nanoprobe was designed for bisphenol A (BPA) determination. It consists of Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) and a DNA-hydrogel technology. Fe3O4 SPIONs were encapsulated in the DNA-hydrogel to form an aggregated state. After adding BPA, the gel system transformed into a sol gel due to the target-aptamer specific binding. The coated gathered particles dispersed and thus, the relaxation time T2 declined. The LNDH nanoprobe was developed to realize a simple, sensitive, and effective BPA determination method without repeated magnetic separation steps. Under the optimal experimental conditions, the determination range of the LNDH biosensor was 10-2~102 ng mL-1 and the limit of determination was 0.07 ng mL-1. The LNDH nanoprobe was applied to two kinds of water samples (tap water and bottled water). The recovery ranged from 87.85 to approximately 97.87%. This strategy offered a new method to detect BPA by LF-NMR. It is also expected to be applicable in related fields of food safety determination, environmental monitoring, and clinical diagnosis. Graphical abstract Schematic presentation of LNDH biosensor. Acrydite-modified ssDNA was copolymerized with acrylamide to form linear conjugates PS-A/B, adding aptamer and SPIONs to form DNA-hydrogel. When aptamer captured the target, the hydrogel was destroyed to disperse the coated SPIONs. T2 relaxation time declined.

[High sensitive ELISA for detection of atrazion].

OBJECTIVES: To set up ELISA for detection of atrazine with high precision. METHODS: The reaction condition of indirect-ELISA was optimized, including atrazine-ovalbumin(AT-OVA) concentration and primary antibody concentration, organic solvent, goat anti-rat immunoglobin G-horseradish peroxidase(IgG-HRP) concentration. The actual samples were detected by the ELISA method established in our laboratory. Then the ELISA method was compared with the HPLC. RESULTS: The specification curve of indirect-ELISA was set up after optimization. The relation coefficient R2=0.9958. The limit of detection (LOD) was 1.972 ng/ml. The percent recovery of the actual samples was in range of 80%~120%. The ELISA detection sensitivity was higher than the HPLC in the range of 0 ng/ml~6 ng/ml atrazine. CONCLUSIONS: The ELISA to detect atrazine has good specificity and high precision. And it can be applied in testing real atrazine samples replacing of the large-scale instrument.

Distribution Characteristics of Spermophilus dauricus in Manchuria City in China in 2015 through '3S' Technology.

Plague is a virulent infectious disease in China. In this study, '3S' technology was used to perform spatial autocorrelation analysis and spatial interpolation analysis for Spermophilus dauricus (S. Dauricus, a species of ground squirrel) captured in Manchuria City in 2015. The results were visually inspected. During the two-month (May to July) plague surveillance in 2015, 198 S. dauricus individuals were captured in the study area in Manchuria City (48 monitoring areas) by using a day-by-day catching method. Spatial autocorrelation was conducted using the ArcGIS software, and the following significantly different results were obtained: Moran's I=0.228472, Z-score=2.889126, and P<0.05. Thus, a spatial aggregation was observed. In 2015, the distribution of S. dauricus diminished from west to east and from north to south of Manchuria. Geo Detector software was used to analyze the habitat factors affecting the spatial distribution of S. dauricus. This highly clustered species mainly exists in suburban communities, construction sites, and areas surrounding factories. In future studies, plague surveillances should be performed in areas around Manchuria and Zhalainuoer.

[Purification of monoclonal antibody to clenbuterol and its biology identity].

OBJECTIVE: To identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection. METHODS: The affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established. RESULTS: The ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml. CONCLUSION: The standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.

[Identification of exotoxin-specific motifs/domains in bacterial exotoxin sequences and corresponding gene ontology analysis].

OBJECTIVE: To identify the exotoxin-specific motifs/domains in bacterial exotoxin sequences, and to expand understanding of bacterial exotoxins pathogenic mechanisms. METHODS: We constructed a non-pathogenic bacterial proteins database and collected 89 bacterial exotoxin sequences from Virulence factor database (VFDB), then we analyzed these protein sequences by motif/domain search using InterProScan (www.ebi.ac.uk/Tools/InterProScan/). RESULTS: We identified 39 exotoxin-specific motifs/domains in 89 bacterial exotoxin sequences. CONCLUSION: The identified exotoxin-specific motifs/domains were closely related to the functions of the exotoxins and could be used as template to search for new exotoxins by mining pathogenic bacterial genomes. The analysis of the acquired Gene Ontology (GO) items was to further expend our understanding of bacterial exotoxin pathogenic mechanisms.

A sensitive immunoassay based on direct hapten coated format and biotin-streptavidin system for the detection of chloramphenicol.

A novel enzyme liked immunosorbent assay (ELISA) was developed for the detection of chloramphenicol (CAP). In this assay, the small molecular hapten (Hap) was directly coated on the surface of microtiter plates and biotin-streptavidin system (BSAS) was employed to improve the sensitivity of immunoassay (BSAS-direct Hap coated ELISA). The surface of microtiter plates was treated with glutaraldehyde (GA) polymer network to introduce aldehyde group, which was used to cross-link with amino group of CAP. Compared with conventional ELISA (the plates were coated with Hap-carrier protein conjugates), the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and HRP-labeled streptavidin were employed in this assay for amplification of signals. The sensitivity of BSAS-direct Hap coated ELISA was increased by approximately 20-folds and the stability was also improved greatly compared to conventional ELISA. Its 50% inhibition concentration (IC(50)) for CAP was 10.5 ng mL(-1) and the limit of detection (LOD) was 0.2 ng mL(-1) after optimization of reaction conditions. To our knowledge, this was one of the most sensitive immunoassay for CAP yet reported. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and high performance liquid chromatography (HPLC). Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.

A fluoroimmunoassay based on quantum dot-streptavidin conjugate for the detection of chlorpyrifos.

A rapid and sensitive competitive fluorescence-linked immunosorbent assay (cFLISA) based on quantum dot-streptavidin conjugate (QDs-SA) was developed for the detection of chlorpyrifos in drinking water. The QDs-SA conjugate, which consists of 3-mercaptopropyl acid-stabilized CdTe nanoparticle QDs and streptavidin (SA) made through the active ester method, was employed to improve the sensitivity of QDs-SA-cFLISA. The 50% inhibition concentration (IC50) and the limit of detection (LOD) were 28.5 and 3.8 ng mL(-1), respectively. QDs-SA-cFLISA increased sensitivity 5.5-fold and reduced detection time by 1 h compared with conventional enzyme-linked immunosorbent assay (ELISA). With chlorpyrifos concentrations of 100, 50, and 20 ng mL(-1), recoveries ranged from 85.9% to 105.3% with coefficients of variation ranging from 6.3% to 13.5%. This study demonstrated that QDs-SA-cFLISA was more rapid and sensitive than conventional ELISA. Therefore, it can be used as a novel screening method for the detection of pesticide residues.

[Preparation of clenbuterol monoclonal antibody with subtractive immunization method].

AIM: To obtain Clenbuterol monoclonal antibodies. METHODS: Clenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique. RESULTS: The mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last. CONCLUSION: Monoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

[Preparation and activity analysis of RGD-mSAK (K130T, K135R)].

In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P(R)P(L) promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5alpha. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68 x 10(5) u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.

[Preparation and the anti-tumor activity of mutant Staphylococcal enterotoxin B].

AIM: To prepare mutant Staphylococcal enterotoxin B(SEB) and observe its anti-tumor activity. METHODS: The expressed mutant SEB-K172E in inclusion body was denatured and renatured, and then isolated and purified. The anti-tumor activity of the mutant SEB-K172E was compared with wild-type SEB. RESULTS: Renaturation and purification method of the mutant SEB-K172E was developed. The anti-tumor activity of acquired mutant protein was ten times higher than that of wild-type SEB. CONCLUSION: The anti-tumor activity of the mutant SEB-K172E is much higher than that of wild-type SEB, suggesting that the mutant SEB-K172E may become a potential anti-tumor drug.