An electrochemical immunosensor based on hybrid self-assembled monolayers for rapid detection of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is highly contagious and poses a serious threat to sericulture production. Because there are currently no effective treatments for BmNPV, a rapid and simple detection method is urgently needed. This paper describes an electrochemical immunosensor for the detection of BmNPV. The immunosensor was fabricated by covalently immobilizing anti-BmNPV, a biorecognition element, onto the surface of the working gold electrode via 11-mercaptoundecanoic acid (MUA)/ß-mercaptoethanol (ME) hybrid self-assembled monolayers. Electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) were used to characterize the electrochemical performance and morphology of the immunosensor, respectively. Under optimum conditions, the developed immunosensor exhibited a linear response to BmNPV polyhedrin in the range of 1 × 102-1 × 108 fg/mL, with a low detection limit of 14.54 fg/mL. The immunosensor also exhibited remarkable repeatability, reproducibility, specificity, accuracy, and regeneration. Normal silkworm blood was mixed with BmNPV polyhedrin and analyzed quantitatively using this sensor, and the recovery was 92.31 %-100.61 %. Additionally, the sensor was used to analyze silkworm blood samples at different time points after BmNPV infection, and an obvious antigen signal was detected at 12 h post infection. Although this result agreed with that provided by the conventional polymerase chain reaction (PCR) method, the electroanalysis method established in this study was simpler, shorter in detection period, and lower in material cost. Furthermore, this innovative electrochemical immunosensor, developed for the ultra-sensitive and rapid detection of BmNPV, can be used for the early detection of virus-infected silkworms.

A molecularly-imprinted SERS sensor based on a TiO2@Ag substrate for the selective capture and sensitive detection of tryptamine in foods.

Herein, a molecularly imprinted surface-enhanced Raman spectroscopy (SERS) sensor was developed for the selective capture and sensitive detection of tryptamine in foods. The SERS sensor exploited silver nanoparticle-decorated TiO2 (TiO2@Ag) substrates for Raman signal enhancement via synergistic effect of electromagnetic enhancement and photoinduced charge-transfer, whilst surface functionalization with the molecularly imprinted polymer ensured selective tryptamine capture. The SERS spectrum of tryptamine on the sensor closely matched that predicted by density functional simulations. The SERS intensity for tryptamine on the developed TiO2@Ag@MIP sensor increased linearly with the logarithm of the tryptamine concentration over the range of 10-6-10-2 mol L-1, with a LOD of 4.85 × 10-7 mol L-1. Tryptamine was detected in a spiked white vinegar sample, and its recoveries were in the range of 92.00%-111.40%. The SERS sensor could be used for the detection of tryptamine in actual samples.

Ultrasensitive determination of sulfathiazole using a molecularly imprinted electrochemical sensor with CuS microflowers as an electron transfer probe and Au@COF for signal amplification.

In this work, a molecularly imprinted sensor employing copper sulfide (CuS) as a novel signal probe was successfully developed for ultrasensitive and selective determination of sulfathiazole (STZ). The reduction signals of Cu2+ produced in the process of electron transfer of CuS containing large amounts of Cu2+ are easy to be captured, which provide high electrochemical signals. Moreover, gold nanoparticles@covalent organic framework with excellent conductivity was introduced on the electrode surface for signal amplification and facilitating electron transfer processes of CuS. Under optimized testing conditions, the proposed sensor offered a linear DPV response to STZ over a very wide concentration range (1.0 × 10-4 to 1.0 × 10-11 mol L-1), with a limit of detection of 4.3 × 10-12 mol L-1. Fodder and mutton samples spiked with STZ were analyzed using this sensor, and the satisfactory recoveries ranging from 83.0% to 107.2% were obtained. In addition, the proposed sensor was used to determine the concentration of STZ in chicken liver and pork liver, with quantification results being near identical to those determined by high-performance liquid chromatography.

Preventive effects of N-acetyl-l-cysteine against imidacloprid intoxication on Bombyx mori larvae.

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N-acetyl-l-cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth-instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth-instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.

Highly Sensitive Electrochemical Determination of Alfatoxin B1 Using Quantum Dots-Assembled Amplification Labels.

A competitive electrochemical immunoassay for highly sensitive detection of AFB1 is demonstrated using layer-by-layer (LBL) assembled quantum dots (QDs) as labels. To investigate the effects of the higher sensitivity of square wave voltammetric stripping (SWV) and of the LBL technique on the proposed immunoassays, the proposed assay was compared to electrochemical (EC) and fluorescent immunoassays, which did not use LBL technology. Peanut samples were analyzed using the three immunoassays. The limits of detection (LODs) were 0.018, 0.046 and 0.212 ng/mL, respectively, while the sensitivities were 0.308, 1.011 and 4.594 ng/mL, respectively. The proposed electrochemical immunoassay displayed a significant improvement in sensitivity, thereby providing a simple and sensitive alternative strategy for determining AFB1 levels in peanut samples.

Functional graphene-gold nano-composite fabricated electrochemical biosensor for direct and rapid detection of bisphenol A.

In this research, the graphene with excellent dispersity is prepared successfully by introducing gold nanoparticle to separate the individual sheets. Various techniques are adopted to characterize the prepared graphene and graphene-gold nanoparticle composite materials. This fabricated new composite material is used as the support material to construct a novel tyrosinase based biosensor for detection of bisphenol A (BPA). The electrochemical performances of the proposed new enzyme biosensor were investigated by differential pulse voltammetry (DPV) method. The proposed biosensor exhibited excellent performance for BPA determination with a wide linear range (2.5×10(-3)-3.0 µM), a highly reproducible response (RSD of 2.7%), low interferences and long-term stability. And more importantly, the calculated detection limit of the proposed biosensor was as low as 1 nM. Compared with other detection methods, this graphene-gold nanoparticle composite based tyrosinase biosensor is proved to be a promising and reliable tool for rapid detection of BPA for on-site analysis of emergency BPA related pollution affairs.

Novel molecularly imprinted stir bar sorptive extraction based on an 8-electrode array for preconcentration of trace exogenous estrogens in meat.

A novel 8-electrode array as stir bar was designed for selective extraction of trace level exogenous estrogens from food samples, followed by liquid desorption and HPLC-photodiode array detection. The array consisted of 8 screen-printed electrodes and each electrode was modified with Fe3O4@meso-/macroporous TiO2 microspheres and molecularly imprinted film (m-TiMIF). The fabrication of the imprinted film coating was very simple without organic solvents and chemical grafting. Both bisphenol A (BPA) and diethylstilbestrol (DES) were employed as templates in m-TiMIF fabrication in order to enrich both targets simultaneously. Interestingly, the imprinted stir bar array showed higher extraction capacity and selectivity for BPA and DES than the non-imprinted counterpart. Meanwhile, it exhibited fast adsorption and desorption kinetics due to increased mass transport in the ultra-thin film. Importantly, the m-TiMIF coating was robust enough for at least 20 uses without obvious alteration in extraction performance. The main parameters affecting the extraction efficiency, including stir speeding, sample pH, ionic strength, extraction time, desorption solvent and time, were optimized. Under optimal experimental conditions, the limits of detection (S/N=3) of the developed method were 0.28 and 0.47 µg L(-1) for BPA and DES respectively, with enrichment factors of 32.6 and 52.8-fold. The linear ranges were 3.0-1500 µg L(-1) and 4.0-1500 µg L(-1) for BPA and DES, respectively. The m-TiMIF-coating conferred better recovery and selectivity, compared with the commercial stir bar coating. The new method was successfully applied to assess BPA and DES in pork and chicken samples with satisfactory recovery.

A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin.

Mulberry leaf is a traditional medicine used to treat diabetes in the clinic. The aim of this study was to determine the mechanisms by which mulberry leaf polysaccharide (MLPII), improves hepatic glucose metabolism and insulin resistance in rats with type 2 diabetes induced by high fat and streptozotocin (STZ). MLPII was administered for 6 weeks after establishment of type 2 diabetes in Wistar rats. At the end of the experiment, oral glucose tolerance, liver glycogen content, glucose synthase (GS) activity and insulin resistance were determined. Expression patterns of proteins and genes associated with insulin signaling as well as biomarkers of oxidative stress and antioxidant enzyme activities were assayed. Compared with normal control rats, MLPII treatment significantly improved oral glucose tolerance (P < 0.01) and restored the glycogen level (P < 0.01) and GS activity (P < 0.05) in diabetic rats. Insulin resistance was improved in MLPII-treated diabetic rats (P < 0.01). Furthermore, expression levels of insulin receptor substrate 2 (IRS2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/AKT) involved in insulin signaling were significantly increased (P < 0.01), while protein­tyrosine phosphatase 1B (PTP1B) expression was markedly reduced (P < 0.01). The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in livers of the MLPII-treated group were significantly reduced (P < 0.01), while activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were significantly increased (P < 0.01, P < 0.01, P < 0.01, respectively). The results clearly indicate that MLPII treatment effectively normalizes hepatic glucose metabolism and insulin signaling by inhibiting the expression of PTP1B, activating the PI3K­AKT pathway and mitigating oxidative stress in the livers of rats with type 2 diabetes induced by high fat and STZ.

Purification, characterization and anti-diabetic activity of a polysaccharide from mulberry leaf.

In the present study, a high-purity polysaccharide from mulberry leaf (MLP) was purified and characterized, and its anti-diabetic effects were investigated in streptozotocin (STZ)-induced diabetic rats. Our results showed that the obtained MLP (purity 99.8%) was determined to be composed of d-arabinose, d-xylose, d-glucose, d-rhamnose and d-mannose with molar ratio of 1:2.13:6.53:1.04:8.73. Oral administration of MLP at 50-200mg/kgbodyweight daily for 5weeks significantly reduced the levels of fasting blood glucose (FBG), glycosylated serum protein (GSP), serum total cholesterol (TC), and serum triglyceride (TG), and increased the body weight, fasting insulin (FINS), C-peptide (C-P), liver glycogen, liver glucokinase, and serum high-density lipoprotein cholesterol (HDL-C). Moreover, MLP promoted marked pancreatic ß-cell regeneration and insulin secretion, and reduced liver fat accumulation in diabetic rats. The treatment effect of MLP on diabetes was similar to the effect of antidiabetic drug glibenclamide. These results clearly indicated that MLP may have a potential for the treatment of hyperglycemia and hyperlipidemia in diabetes.

Anti-diabetic effect of mulberry leaf polysaccharide by inhibiting pancreatic islet cell apoptosis and ameliorating insulin secretory capacity in diabetic rats.

Diabetes mellitus is a clinically complex disease characterized by chronic hyperglycemia with metabolic disturbances. In this study, we investigated the effect of mulberry leaf polysaccharide (MLPII) on pancreatic islet cell apoptosis and insulin secretory function in diabetic rats induced by a high fat diet and streptozotocin. Our results showed that MLPII treatment inhibited pancreatic islet cell apoptosis and ameliorated insulin secretory capacity of pancreatic ß-cells in diabetic rats. And further study demonstrated that chronic treatment of diabetic rats with MLPII resulted in up-regulation of anti-apoptotic B-cell leukaemia/lymphoma 2 (Bcl-2) protein and down-regulation of pro-apoptotic Bcl2-associated X (Bax) and caspase-3 protein in pancreatic islet cells. Moreover, MLPII significantly restored pancreatic duodenal homeobox-1 (PDX-1) protein nuclear localization, and increased mRNA and protein expression of PDX-1 and its downstream targets, glucose transporter 2 (GLUT2) and glucokinase (GCK) in pancreatic islet cells of diabetic rats. These findings suggested that MLPII might play a critical role in protecting pancreatic islet cell from apoptosis via elevation of Bcl-2/Bax ratio, and ameliorating insulin secretory capacity of pancreatic ß-cells via restoration of PDX-1 nuclear localization and expression levels in diabetic rats. This is the first report to explore the potential molecular mechanism involved in the hypoglycemic activity of the polysaccharide from mulberry leaves.

Media optimization for laccase production by Trichoderma harzianum ZF-2 using response surface methodology.

Trichoderma harzianum ZF-2 producing laccase was isolated from decaying samples from Shandong, China, and showed dye decolorization activities. The objective of this study was to optimize its culture conditions using a statistical analysis of its laccase production. The interactions between different fermentation parameters for laccase production were characterized using a Plackett-Burman design and the response surface methodology. The different media components were initially optimized using the conventional one-factor-at-a-time method and an orthogonal test design, and a Plackett-Burman experiment was then performed to evaluate the effects on laccase production. Wheat straw powder, soybean meal, and CuSO4 were all found to have a significant influence on laccase production, and the optimal concentrations of these three factors were then sequentially investigated using the response surface methodology with a central composite design. The resulting optimal medium components for laccase production were determined as follows: wheat straw powder 7.63 g/l, soybean meal 23.07 g/l, (NH4)2SO4 1 g/l, CuSO4 0.51 g/l, Tween-20 1 g/l, MgSO4 1 g/l, and KH2PO4 0.6 g/l. Using this optimized fermentation method, the yield of laccase was increased 59.68 times to 67.258 U/ml compared with the laccase production with an unoptimized medium. This is the first report on the statistical optimization of laccase production by Trichoderma harzianum ZF-2.

[Control effect and bacteriostasis of mulberry endophytic bacterium Burkholderia cepacia Lu10-1 on silkworm septicemia].

A laboratory test was conducted to study the control effect and bacteriostasis of antagonistic bacterium Burkholderia cepacia Lu10-1 isolated from mulberry on silkworm septicemia, aimed to develop a new microbial pesticide to control silkworm diseases. The supernatant of Lu10-1 zymotic fluid achieved 41.2% control efficiency and 24.0% prophylactic effect on silkworm septicemia. The antibacterial crude extract of Lu10-1 had stronger antagonistic activity against Bacillus bombyseptieus. The diameter of inhibition zone reached 18.20 mm, and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the antibacterial crude extract were 1.56 and 3.13 mg x mL(-1), respectively. After treated with the antibacterial crude extract, B. bombyseptieus never appeared logarithmic growth phase, its cell membrane permeability changed, intracellular protein leaked out, intracellular macromolecular protein degraded, and at last, the thalli cracked, inner substances out-flowed, cavity formed, and cell ablated. It was considered that the antagonistic substances of Lu10-1 strain could be used for controlling silkworm septicemia, with preferable development foreground.

[Identification and colonization of an antagonistic endophytic Burkholderia cepacia Lu10-1 isolated from mulberry].

OBJECTIVE: To identify and colonize an antagonistic bacterium, Lu10-1, isolated from the healthy mulberry. METHODS: Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. A spontaneous Lu10-1 mutant tolerant to rifampicin and ampicillin were isolated by gradually increasing the concentration of the two antibiotics. The mutants were used to assess the ability of Lu10-1 to colonize mulberry by different inoculation approaches, including stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. RESULTS: Lu10-1 belonged to Burkholderia. In the phylogenetic tree, Lu10-1 was the closest relative to B. cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Moreover, our results also indicated that the population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed soaking. The bacterial density inside the mulberry seedling tissues decreased to a steady level 20 days after germination. The population of strain Lu10-1 in mulberry leaves and stems after the treatment of root soaking increased first and then decreased. CONCLUSION: The strain Lu10-1 fell into Burkholderia cepacia genomovar I as a single species. Furthermore, the strain Lu10-1 could colonize and transmit in mulberry, while its resistance to plant pathogen was not changed during the process of colonization compared to the original strains. Taken together, we suggest that Burkholderia. cepacia Lu10-1 will play an important role in the biological control of mulberry disease.