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Li L.,Jimei University | Peng A.-H.,Jimei University | Lin Z.-Z.,Jimei University | Zhong H.-P.,Jimei University | And 3 more authors.
Food Chemistry | Year: 2017

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31 μg L−1 and the detection limit being 0.3 μg L−1. The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately. © 2017 Elsevier Ltd


Wu L.,Jimei University | Lin Z.-Z.,Jimei University | Zhong H.-P.,Jimei University | Peng A.-H.,Jimei University | And 3 more authors.
Food Chemistry | Year: 2017

A sensitive fluorescence sensor for the detection of malachite green (MG) was fabricated by grafting molecularly imprinted polymers (MIPs) onto the surface of CdTe quantum dots (QDs). The MIP-coated QDs were synthesized via a reverse microemulsion method using (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as functional monomer and cross-linker, respectively. The optimum molar ratio of MG, functional monomer and cross-linker was 1:3:10. The MIP-coated QDs exhibited uniform spheres with diameter around 49 nm and excellent fluorescence emission at λex 370 nm. A linear relationship with two segments between the relative fluorescence intensities and the MG concentrations ranging from 0.08 to 20 μmol·L−1 could be obtained with a detection limit of 12 μg·kg−1. The fluorescent probe was successfully applied to the determination of MG in fish samples with the spiked recoveries ranging from 94.3% to 109.5% which were in accordance with those of the measurement by HPLC-UV. © 2017 Elsevier Ltd


Mao K.,State Oceanic Administration | Mao K.,Xiamen University | Chen W.,State Oceanic Administration | Chen W.,Xiamen University | And 5 more authors.
Fish and Shellfish Immunology | Year: 2017

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9–58.4% identity to other teleost fish CD4-1 molecules, and 16.4–20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish. © 2017 Elsevier Ltd


Lin Z.,Jimei University | Wang D.,Jimei University | Peng A.,Jimei University | Huang Z.,Jimei University | Huang Z.,Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources
International Journal of Polymer Analysis and Characterization | Year: 2017

A magnetic molecularly imprinting polymer for domoic acid was fabricated. Synthesis conditions were optimized. The polymer particles have high magnetization for rapid magnetic separation. The apparent maximum absorption amount and dissociation constant of the polymer were 1,600 µg g−1 and 20.6 µg mL−1, respectively. The polymer retained 90% of adsorption amount after 5 times of repeated use. It was used as an adsorbent for purification and HPLC detection of domoic acid in shellfish with a detection limit of 0.050 µg g−1. Thus, the polymer could be applied to the sample pretreatment of aquatic products. © 2017 Taylor & Francis


Wu L.,Jimei University | Lin Z.-Z.,Jimei University | Zhong H.-P.,Jimei University | Chen X.-M.,Jimei University | And 2 more authors.
Sensors and Actuators, B: Chemical | Year: 2017

A fluorescent probe based on CdTe quantum dots (QDs) coated with molecularly imprinted polymers (MIPs) was fabricated for the determination of malachite green (MG) by the strategy of fluorescence resonance energy transfer (FRET). The MIP-coated QDs were synthesized through precipitation polymerization using acrylamide and ethylene glycol dimethacrylate as monomer and cross-linker, respectively. The optimum molar ratio of MG, monomer and cross-linker was 1:8:40 in 60 mL of acetonitrile, and the polymerization time was 28 h. The MIP-coated QDs, with an average diameter around 290 nm, showed excellent fluorescence emission in 500–700 nm at λex 370 nm. The probe exhibited fluorescence quenching response to MG selectively within only 5 min at the concentrations from 0.1 to 20 μmol L−1. The fluorescent probe was successfully used to detect MG in water and fish samples with a detection limit of 0.059 μmol L−1 (3σ, n = 9). The spiked recoveries, from 94.8% to 98.1% for water samples, and 98.1% to 106.2% for fish samples, respectively, indicated that the as-prepared MIP-coated QDs could be used as a fluorescent probe to detect MG rapidly in water and fish samples. © 2016 Elsevier B.V.


Li M.,State Oceanic Administration | Li M.,Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources | Guo W.,State Oceanic Administration | Guo W.,Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2016

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X4-Cys-X2-Cys-X29-35-Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors. © 2016 Springer-Verlag Berlin Heidelberg


Shu L.,Xiamen University | Yang Y.,Xiamen University | Huang H.,Xiamen University | Ye H.,Xiamen University | Ye H.,Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources
Molecular and Cellular Endocrinology | Year: 2016

In vertebrates, bone morphogenetic proteins (BMPs) play an important role in various biological processes. However, the function of BMPs in crustaceans is still unknown. In our study, a ligand (BMP7) and two receptors (Sp-BMPRIB and Sp-BMPRII) are cloned firstly in the mud crab, Scylla paramamosain. The qRT-PCR demonstrated that both ligand and receptors were expressed in various tissues, especially in ovary. The expression of BMPRs mRNA increased along the ovarian development, while BMP7 had an opposite tendency. In-situ hybridization revealed that Sp-BMPRIB and Sp-BMPRII were expressed in both oocytes and follicle cells, whereas Sp-BMP7 was exclusively localized in follicle cells. RNAi experiments showed that the expression levels of Smad1 and vitellogenin receptor declined rapidly after BMPRs were silenced. Based on these data, we hypothesized that in S. paramamosain, BMP7 and BMPRs had impact on the ovarian development, presumably via the autocrine/paracrine way. © 2016 Elsevier Ireland Ltd


Lin Z.,Jimei University | Lin Z.,Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering | Wang D.,Jimei University | Peng A.,Jimei University | And 4 more authors.
Journal of Separation Science | Year: 2016

A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5-pentanetricarboxylic acid as the template molecule, 4-vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween-80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 μg/mL and apparent maximum adsorption capacity of 1249 μg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high-performance liquid chromatography. The detection limit of 0.17 μg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0–98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Li L.,Jimei University | Lin Z.-Z.,Jimei University | Peng A.-H.,Jimei University | Zhong H.-P.,Jimei University | And 3 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2016

A direct competitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection of malachite green (MG) with a high sensitivity and selectivity using magnetic molecularly imprinted polymers (MMIPs) as a bionic antibody. MMIPs were prepared through emulsion polymerization using Fe3O4 nanoparticles as magnetic nuclei, MG as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and span-80/tween-80 as mixed emulsifiers. The MMIPs were characterized by scanning electron micrographs (SEM), thermal-gravimetric analyzer (TGA), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM), respectively. A high magnetic saturation value of 54.1 emu g−1 was obtained, resulting in rapid magnetic separation of MMIPs with an external magnet. The IC50 of the established ELISA method was 20.1 μg L−1 and the detection limit (based on IC85) was 0.1 μg L−1. The MMIPs exhibited high selective binding capacity for MG with cross-reactivities less than 3.9% for MG structural analogues. The MG spiking recoveries were 85.0%–106% with the relative standard deviations less than 4.7%. The results showed that the biomimetic ELISA method by using MMIPs as bionic antibody could be used to detect MG rapidly in fish samples with a high sensitivity and accuracy. © 2016 Elsevier B.V.


Zhang Y.,State Oceanic Administration | Zhang Y.,Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources | Chen D.,China National Center for Food Safety Risk Assessment | Hong Z.,State Oceanic Administration | Hong Z.,China National Center for Food Safety Risk Assessment
Toxins | Year: 2015

In this study, we developed a self-assembly pipette tip solid-phase extraction (PTSPE) method using a high molecular weight polymer material (PAX) as the adsorbent for the determination of domoic acid (DA) in human urine samples by liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis. The PTSPE cartridge, assembled by packing 9.1 mg of PAX as sorbent into a 200 μL pipette tip, showed high adsorption capacity for DA owing to the strong cationic properties of PAX. Compared with conventional SPE, the PTSPE is simple and fast, and shows some advantages in the aspects of less solvent consumption, low cost, the absence of the evaporation step, and short time requirement. All the parameters influencing the extraction efficiency such as pH, the amount of sorbent, the number of aspirating/dispensing cycles, and the type and volume of eluent in PTSPE were carefully investigated and optimized. Under the optimized conditions, the limit of detection (LOD) and limit of quantification (LOQ) values of DA were 0.12 μg/L and 0.37 μg/L respectively. The extraction recoveries of DA from the urine samples spiked at four different concentrations were in a range from 88.4% to 102.5%. The intra- and inter-day precisions varied from 2.1% to 7.6% and from 2.6% to 12.7%, respectively. The accuracy ranged from -1.9% to -7.4%. © 2015 by the authors; licensee MDPI, Basel, Switzerland.

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