Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification.

Objective: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) . Methods: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD). Results: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g. Conclusion: The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Amorphous poly-N-vinylcarbazole polymer as a novel matrix for the determination of low molecular weight compounds by MALDI-TOF MS.

Traditional matrices for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) are usually crystalline small molecules. The heterogeneous co-crystallization of the analyte and the matrix creates a sweet spot effect and reduces point-to-point reproducibility. In this study, an amorphous poly-N-vinylcarbazole polymer (PVK) was studied as a novel matrix for MALDI-TOF MS to detect various low molecular weight compounds (LMWCs) in the negative ion mode. The PVK achieved excellent matrix action and showed high sensitivity, good salt tolerance, and reproducibility. These results significantly broaden the design rules for new and efficient polymeric MALDI matrices.

Fabrication of Magnetic Al-Based Fe3O4@MIL-53 Metal Organic Framework for Capture of Multi-Pollutants Residue in Milk Followed by HPLC-UV.

The efficient capture of multi-pollutant residues in food is vital for food safety monitoring. In this study, in-situ-fabricated magnetic MIL-53(Al) metal organic frameworks (MOFs), with good magnetic responsiveness, were synthesized and applied for the magnetic solid-phase extraction (MSPE) of chloramphenicol, bisphenol A, estradiol, and diethylstilbestrol. Terephthalic acid (H2BDC) organic ligands were pre-coupled on the surface of amino-Fe3O4 composites (H2BDC@Fe3O4). Fe3O4@MIL-53(Al) MOF was fabricated by in-situ hydrothermal polymerization of H2BDC, Al (NO3)3, and H2BDC@Fe3O4. This approach highly increased the stability of the material. The magnetic Fe3O4@MIL-53(Al) MOF-based MSPE was combined with high-performance liquid chromatography-photo diode array detection, to establish a novel sensitive method for analyzing multi-pollutant residues in milk. This method showed good linear correlations, in the range of 0.05-5.00 µg/mL, with good reproducibility. The limit of detection was 0.004-0.108 µg/mL. The presented method was verified using a milk sample, spiked with four pollutants, which enabled high-throughput detection and the accuracies of 88.17-107.58% confirmed its applicability, in real sample analysis.

The Role of Suspension Array Technology in Rapid Detection of Foodborne Pollutants: Applications and Future Challenges.

Food safety is an important livelihood issue, which has always been focused attention by countries and governments all over the world. As food supply chains are becoming global, food quality control is essential for consumer protection as well as for the food industry. In recent years, a great part of food analysis is carried out using new techniques for rapid detection. As the first biochip technology that has been approved by the Food and Drug Administration (FDA), there is an increasing interest in suspension array technology (SAT) for food and environmental analysis with advantages of rapidity, high accuracy, sensitivity, and throughput. Therefore, it is important for researchers to understand the development and application of this technology in food industry. Herein, we summarized the principle and composition of SAT and its application in food safety monitoring. The utility of SAT in detection of foodborne microorganisms, residues of agricultural and veterinary drugs, genetically modified food and allergens in recent years is elaborated, and the further development direction of SAT is envisaged.

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.

Suspension array for multiplex immunoassay of five common endocrine disrupter chemicals.

A low cost and effective indirect competitive method is reported to detect five EDCs, 17-beta-estradiol (E2), estriol (E3), bisphenol A (BPA), diethylstilbestrol (DES), and nonylphenol (NP) simultaneously, based on suspension array technology (SAT). Five kinds of complete antigens (E2-BSA, E3-BSA, BPA-BSA, DES-BPA, NP-BSA) were coupled to different encoding microspheres using purpose-made solutions in our laboratory instead of commercially available amino coupling kits; the method was further optimized for determination and reducing  the cost. Encoding and signaling fluorescence of the particles are determined at 635/532 nm emission wavelengths. High-throughput curves of five EDCs were draw and the limit of detection (LOD) were between 0.0010 ng mL-1 ~ 0.0070 ng mL-1. Compared with traditional ELISA methods, the SAT exhibited better specificity and sensitivity. Experiments using spiked milk and tap water samples were also carried out, and the recovery was between 85 and 110%; the results also confirmed good repeatability and reproducibility. It illustrated great potential of the present strategy in the detection of EDCs in actual samples.

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.

Development and application of magnetic solid phase extraction in tandem with liquid-liquid extraction method for determination of four tetracyclines by HPLC with UV detection.

A novel HPLC-UV method was developed for the determination of four tetracyclines based on magnetic solid phase extraction in tandem with liquid-liquid extraction. The water-soluble amino functionalized magnetite nanoparticle (MNP-NH2) was used as an adsorbent for extraction/preconcentration of tetracycline, oxytetracycline, chlortetracycline, and doxycycline from bovine milk samples. Fourier transform infrared spectrometer, transmission electron microscope, X-ray diffraction, and elemental analyze techniques were used to characterize the material. Some key parameters which influence liquid-liquid extraction and magnetic dispersive solid-phase extraction procedure, including volume of extraction solvent, the amount of adsorbent, the pH, extraction and desorption time, the composition of the eluent, and elution frequency were investigated. The proposed method exhibited a linear range of 50.0-2500.0 µg L-1 (r2 = 0.9941) with and good reproducibility (RSD < 2.2%, n = 3). The limit of detection and quantification were 40.0 and 50.0 µg L-1. This method was verified using milk sample spiked with four tetracyclines (100.0-200.0 µg L-1), and accuracies of 87.8-107.5%, which confirmed its applicability in real-sample analysis. The proposed method also shows potential application prospects for other water-soluble adsorbents.

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.

Low field nuclear magnetic sensing technology based on hydrogel-coated superparamagnetic particles.

Based on superparamagnetic nanoparticles, a responsive polyacrylamide hydrogel self-assembled by nucleic acid hairpin hybridization chain reaction was designed, and a universal low field nuclear magnetic resonance sensing platform was successfully constructed. As the target was gradually added, the hydrogel coating on the surface of the magnetic nanoparticle was opened layer by layer through binding with the aptamer, which specifically bonded thereto, causing different degrees of exposure of the magnetic nanoparticle, resulting in changes of low field nuclear magnetic resonance signals. This method was originally applied to the rapid detection of adenosine triphosphate (ATP), and the versatility of the method was verified using polychlorinated biphenyl 77 (PCB77). This method had the advantage of being fast, convenient, and low cost, and it can be easily operated with high repeatability. This universal method can detect a variety of targets by replacing aptamers and may be useful in controlling food quality and for rapidly detecting cancer cells in vitro.

Graphene oxide composites for magnetic solid-phase extraction of twelve quinolones in water samples followed by MALDI-TOF MS.

Antibiotic compounds in natural waters are normally present at low concentrations. In this paper, an easy and highly sensitive screening method using graphene oxide-functionalized magnetic composites (GO@NH2@Fe3O4) combined with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was established for twelve quinolone antibiotics. GO@NH2@Fe3O4 composites were utilized as adsorbents for magnetic solid-phase extraction. This method combines the advantages of magnetic solid-phase extraction and MALDI-TOF MS, which allows for fast detection of quinolones at low concentrations. To improve absorption efficiency, the following parameters were individually optimized: sample acidity, extraction time, amount of adsorbent used, eluent used, and desorption time. Under the optimum conditions, the established method gave a low detection limit of 0.010 mg/L and allowed the high-throughput screening of twelve quinolone antibiotics (enoxacin, norfloxacin, ciprofloxacin, pefloxacin, fleroxacin, gatifloxacin, enrofloxacin, levofloxacin, sparfloxacin, danofloxacin, difloxacin, and lomefloxacin). The proposed method, having an easily prepared sorbent with a high affinity for quinolones and a convenient, high-throughput detection step, has been shown to have merit for the detection of antibiotics in water samples. Graphical abstract Schematic illustration of the (A) preparation of GO@NH2@Fe3O4 and (B) operating procedure for the MSPE and MALDI-TOF MS detection of QNs.

[Effects of soybean isoflavones on the energy metabolism of swimming mice].

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(P<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(P<0.05). CONCLUSIONS: Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.

[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.

Microarray expression profiling and co-expression network analysis of circulating LncRNAs and mRNAs associated with neurotoxicity induced by BPA.

A growing body of evidence has shown bisphenol A (BPA), an estrogen-like industrial chemical, has adverse effects on the nervous system. In this study, we investigated the transcriptional behavior of long non-coding RNAs (lncRNAs) and mRNAs to provide the information to explore neurotoxic effects induced by BPA. By microarray expression profiling, we discovered 151 differentially expressed lncRNAs and 794 differentially expressed mRNAs in the BPA intervention group compared with the control group. Gene ontology analysis indicated the differentially expressed mRNAs were mainly involved in fundamental metabolic processes and physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and neuroinflammation responses. In the expression network of the BPA-induced group, a great number of nodes and connections were found in comparison to the control-derived network. We identified lncRNAs that were aberrantly expressed in the BPA group, among which, growth arrest specific 5 (GAS5) might participate in the BPA-induced neurotoxicity by regulating Jun, RAS, and other pathways indirectly through these differentially expressed genes. This study provides the first investigation of genome-wide lncRNA expression and correlation between lncRNA and mRNA expression in the BPA-induced neurotoxicity. Our results suggest that the elevated expression of lncRNAs is a major biomarker in the neurotoxicity induced by BPA.

Synthesis and Characteristics of Large-Area and High-Filling CdS Nanowire Arrays in AAO Template.

CdS nanowires arrays were successfully synthesized by a simple solvothermal process using AAO as templates. The phase structures, morphologies, and optical properties of the products were investigated by X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, Raman spectroscopy, and photoluminescence spectroscopy. It was found that the nanowires were composed of hexagonal structure CdS nanoparticles and the average diameters is about 60-70 nm. A strong green emission with a maximum around 505 nm was observed from the synthesized CdS nanowires at room temperature, which was attributed to near-band-edge emission. A 3D self-seed nucleation coalescent process was proposed for the formation of CdS nanowires structures. The present synthetic route is expected to be applied to the synthesis of other II-VI groups or other group's 1D semiconducting materials.

Clinical characteristics associated with the intracranial dissemination of gliomas.

OBJECTIVES: Glioma is the most common malignant tumor of the brain and the intracranial dissemination of gliomas is the late stage of the development of the tumor. However, there is little research in literature on the occurrence of intracranial dissemination of gliomas. In order to provide a reference for clinical work, we carried out this study on intracranial dissemination of glioma. PATIENTS AND METHODS: A total of 629 patients with gliomas received tumor resection by the same surgeon from August 2010 to September 2015 were included in this study. The authors performed a retrospective review of the patients and the information regarding clinical features, histopathological results, molecular pathologic results and clinical outcomes was collected and analyzed. RESULTS: In this retrospective study, we found that the intracranial dissemination phenomenon occurred in 53 patients (8.43%). We analyzed the clinical characteristics of patients and found that the age at diagnosis (P = 0.011), WHO grade of the tumor (P < 0.001), and involvement of the corpus callosum (P = 0.010) were associated with the occurrence of dissemination. The higher grade of the tumor, the more prone to disseminate. Deletion of 1p/19q had no significant correlation with the intracranial dissemination. MMP9, Ki-67, and EGFR were highly expressed in tumor cells that caused dissemination, and the level of Ki-67 expression had significance in statistics (P < 0.01). CONCLUSION: In our study, older age (>40 years), high pathological grade, invasion of the corpus callosum and high levels of Ki-67 expression were risk factors associated with the intracranial dissemination of gliomas.

[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.

Investigating the quantitative structure-activity relationships for antibody recognition of two immunoassays for polycyclic aromatic hydrocarbons by multiple regression methods.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants found in the environment. Immunoassays represent useful analytical methods to complement traditional analytical procedures for PAHs. Cross-reactivity (CR) is a very useful character to evaluate the extent of cross-reaction of a cross-reactant in immunoreactions and immunoassays. The quantitative relationships between the molecular properties and the CR of PAHs were established by stepwise multiple linear regression, principal component regression and partial least square regression, using the data of two commercial enzyme-linked immunosorbent assay (ELISA) kits. The objective is to find the most important molecular properties that affect the CR, and predict the CR by multiple regression methods. The results show that the physicochemical, electronic and topological properties of the PAH molecules have an integrated effect on the CR properties for the two ELISAs, among which molar solubility (S(m)) and valence molecular connectivity index ((3)χ(v)) are the most important factors. The obtained regression equations for Ris(C) kit are all statistically significant (p < 0.005) and show satisfactory ability for predicting CR values, while equations for RaPID kit are all not significant (p > 0.05) and not suitable for predicting. It is probably because that the Ris(C) immunoassay employs a monoclonal antibody, while the RaPID kit is based on polyclonal antibody. Considering the important effect of solubility on the CR values, cross-reaction potential (CRP) is calculated and used as a complement of CR for evaluation of cross-reactions in immunoassays. Only the compounds with both high CR and high CRP can cause intense cross-reactions in immunoassays.