High-throughput, high-level production of PS-tag-fused single-chain Fvs by microplate-based culture

In the present study, we investigated a microplate-based culture (MBC) of Escherichia coli for the high-throughput, high-level production of PS-tag-fused scFvs (scFv-PS) in insoluble form. The Overnight Express™ Autoinduction System (OE system) was adopted to skip the laborious induction step of the addition of IPTG. ScFv and scFv-PS began to be expressed after 6 h by conventional flask culture (FLC) and by MBC when utilizing the OE system, and similar specific productivity levels were attained during cultivation. In MBC, an important factor that directly affected the production levels was rotational speed during cultivation, suggesting that the mass transfer of oxygen was rate-limiting. In a comparison of the productivity of flask cultures utilizing the 2YT-IPTG and OE systems, MBC utilizing the OE system was the highest, with approximately 1 mg of insoluble scFv-PS obtained from each well under optimal conditions (1400 rpm). The results of SDS-PAGE and a cross-contamination check indicated that very similar cultivation conditions were attained in each well, without cross-contamination. Thus, MBC using the OE system is very useful for the high-throughput, high-level production of scFv-PS, which can be activated on the surface of hydrophilic PS plates by solid-phase refolding. Therefore, the production of a variety of specific scFv-PSs for cytokines and biomarkers will make possible the construction of sensitive and low-cost antibody microarrays, which will be very useful in clinical diagnosis and biochemical research.     

用溶氧仪代替碘量法测定生化需氧量中的溶解氧

采用溶氧仪代替碘量法测定水质生化需氧量中的溶解氧，讨论了测定中注意事项。经两种方法检测数据对比和标准物质回收试验，表明该测定方法完全能满足日常检测工作需要，既简化了测定程序，又节省了化学试剂。     

Organic acids,biochemical oxygen demand and chemical oxygen demand in the soluble fraction of piggery slurry

Close, largely linear, relationships were found between volatile fatty acids (VFA) determined by gas chromatography, total organic acids (TOA) determined colorimetrically, biochemical oxygen demand (BOD₅) and chemical oxygen demand (COD) in the soluble fraction of piggery slurries. Samples were taken from untreated and aerobically-treated slurries. All correlations were significant at P=0.001, with values of r ranging between 0.88 for the correlation of VFA and COD, to 0.97 for the correlation of TOA and BOD₅. VFA accounted for up to 69% of BOD₅ and 55% of COD. It is suggested that organic acids could replace soluble BOD₅ in some situations, since the correlations were good. Prediction of COD from organic acids, or BOD₅ from COD, were less accurate, particularly when concentrations were low, since COD contains a biologically inert fraction, which varies with storage. Soluble organic nitrogen was not useful in predicting BOD₅ or COD, correlating only at P=0.02.     

High-throughput determination of citrinin in rice by ultra-high-performance liquid chromatography and fluorescence detection (UHPLC-FL)

A rapid, simple and effective method for the determination of citrinin in rice by UHPLC coupled to fluorescence detection has been developed and validated. Extraction of citrinin from rice samples was achieved by applying a QuEChERS-based extraction/partitioning process with water and acetonitrile, without the need of further extract clean-up. The method was fully validated for white rice, and its applicability in other rice matrices, such as brown and red rice, was confirmed by recovery experiments. Under optimum conditions, recoveries ranged from 72.5% to 92.8%, with relative standard deviations (RSDs) lower than 7.1%. Detection and quantification limits were estimated to be 1.5 and 5.0 µg kg^?1, respectively. Finally, 21 organic rice samples were analysed, but none was contaminated with citrinin above the detection limit of the method. The method proved to be fast and non-laborious.     

Estimation of beer stability by sulphur dioxide and polyphenol determination. Evaluation of a Laccase-Sonogel-Carbon biosensor

The traditional methods used to evaluate parameters related to beer ageing have been tested, revised and criticised. With this aim, a sampling of lager beers has been carried out. Sulphur dioxide, polyphenols and antioxidant capacity have been determined in fresh and aged beer. Only sulphur dioxide concentration showed relevant differences after the ageing process. These results together with a correlation study provided information about the selectivity of the methodologies used and have made it clear that sulphur dioxide has an important role and polyphenols have an indirect role. Laccase-Sonogel-Carbon biosensor is proposed to determine polyphenols in beers instead of Folin–Ciocalteu assay due to its better selectivity, sensitivity and short assay times.     

利用SPR生物传感器检测盐酸克伦特罗

目前畜牧业生产中盐酸克伦特罗的使用屡禁不止,因此为了有效、准确的检测动物源性产品中药物残留量,各种检测方法也随之在逐渐进步与完善。其中SPR生物传感器检测法作为一种新型的检测药物残留的方法,与其它检测方法相比,有着样品无需标记、实时监测反应的动态过程、灵敏度高、检测时间短、效率高、无损伤检测等优点,其主要检测方法有直接法、抑制法、竞争法。虽然SPR生物传感器目前还没有得到广泛推广使用,随着抗体技术不断进步,理论及方法的逐渐完善,必将促使SPR生物传感器在食品安全分析和药残检测方面得到广泛应用。     

Cyanide determination in fruit brandies by an amperometric biosensor with immobilised Saccharomyces cerevisiae

A microbial sensor consisting of immobilised Saccharomyces cerevisiae and an oxygen electrode was applied to determine cyanide content in samples of fruit brandies. The results obtained by biosensor measurements were compared by ISE-potentiometry. The average recovery was 97.8% with standard deviation of 6.8%. This microbial sensor was reused for hours daily (strongly depending on the determined total cyanide concentration).     

高效液相色谱法快速测定植物油中苯并(a)芘含量

采用乙腈饱和正己烷除去植物油中大部分油脂,并用正己烷饱和乙腈数次提取苯并芘,采用HPLC系统在流动相为乙腈∶水(90∶10)、荧光检测器:激发波长384 nm,发射波长406 nm下检测,用外标法峰面积定量。结果显示:苯并芘在4.0~80.0 ng/mL浓度范围内峰面积与浓度线性关系良好,加标回收率为92.7%~98.2%,方法定量限为2μg/kg。该法简便易行、准确可靠。     

多维气相色谱/质谱法快速测定卷烟烟气中的苯并(a)芘

自行组装了一套具有全自动和在线富集功能的大孔径-毛细柱多维气相色谱(MDGC)系统。应用该系统,开发了一种简便快速且准确的检测卷烟烟气苯并(a)芘的方法。收集在剑桥滤片上的烟气冷凝物用环己烷提取和浓缩后,不需要任何净化,可直接进行MDGC-MS-SIM分析。方法的重现性、回收率和检测限均令人满意。对不同配方和过滤嘴的卷烟样品进行了定量测定,对样品的测定结果进行了比较。      

Investigation of a multienzyme based amperometric biosensor for determination of sucrose in fruit juices

Determination of sucrose content is one of the most important routinely performed tests for quality control in the beverage industry. Thus, it is rather important to have a fast, simple and sensitive method that can be used for determination of sucrose in sweet-drink processing. The biosensor investigated was based on a thin layer enzyme cell in which glucose oxidase, invertase and mutarotase were immobilized on a protein membrane. Hydrogen peroxide as the product of the consecutive enzyme reaction was detected by amperometric technique. The optimum enzyme composition of invertase, mutarotase, and glucose oxidase for immobilisation was found to be 8498, 672, and 120 U using 2.2 mg BSA as protective agent dissolved in 200 μL phosphate buffer solution. To achieve the best measuring technique, changes in several parameters such as pH value, temperature and flow rate were studied. Having found the optimum conditions, the statistical parameters of the analysis were established. The linear range of the determination was between 1.0 × 10⁻⁴ and 2.5 × 10⁻³ mol/L with a correlation coefficient of 0.99. The sucrose content of several fruit juices was determined with this sensor, and the results were compared with an enzymatic reference method. The correlation between the two sucrose determination methods was found to be 0.97.     

L-抗坏血酸传感器在果汁中Vc含量的测定

本文利用花椰菜组织通过交联制备L-抗坏血酸传感器,该传感器在pH6.0、0.1mol/L的KH_2PO_4-Na_2HPO_4真缓冲溶液中对Vc标准溶液的线性范围为5.0×10~(-5)×1.8×10~(-3)mol/L,响应时间1min。电极使用寿命在1个月以上。利用该传感器测定了部分水果和果汁中Vc的含量,所得结果与分光光度法(2,4-二硝基苯肼法)测得结果一致。     

Development of chemical oxygen demand on-line monitoring system based on a photoelectrochemical degradation principle

A simple, rapid, and sensitive on-line chemical oxygen demand (COD) determination method has been proposed and experimentally validated. The method is based on a photoelectrochemical oxidative degradation principle and operates under a continuous flow mode. The method employs a specially designed thin-layer photoelectrochemical cell that incorporates a highly effective nanoparticulate TiO2 photoanode. This approach overcomes many problems associated with the conventional COD determination techniques such as long analysis time, consumption of expensive and toxic reagents, production of secondary toxic waste, and poor reproducibility. The effect of important experimental parameters on the analytical signal generation was systematically investigated, and the optimum conditions were obtained. The method was successfully applied to determine the COD of real samples from various industrial wastewaters. The COD value of real samples determined by this method agreed well with the standard dichromate method. The assay time of 1-5 min/sample can be readily achieved. A practical detection limit of 1 mg L(-1) COD with a linear range of 1-100 mg L(-1) was achieved under the optimum conditions.     

High-throughput method for the determination of residues of β-lactam antibiotics in bovine milk by LC-MS/MS

This study describes the development and validation procedures for scope extension of a method for the determination of β-lactam antibiotic residues (ampicillin, amoxicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, ceftiofur, cefquinome, cefoperazone, cephapirine, cefalexin and cephalonium) in bovine milk. Sample preparation was performed by liquid-liquid extraction (LLE) followed by two clean-up steps, including low temperature purification (LTP) and a solid phase dispersion clean-up. Extracts were analysed using a liquid chromatography-electrospray-tandem mass spectrometry system (LC-ESI-MS/MS). Chromatographic separation was performed in a C18 column, using methanol and water (both with 0.1% of formic acid) as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Main validation parameters such as linearity, limit of detection, decision limit (CCα), detection capability (CCβ), accuracy, and repeatability were determined and were shown to be adequate. The method was applied to real samples (more than 250) and two milk samples had levels above maximum residues limits (MRLs) for cloxacillin – CLX and cefapirin – CFAP.     

基于高通量测序的紫贻贝中微生物群落分析

为探究不同状态下紫贻贝制品表面附着的微生物菌群组成情况。本实验以新鲜紫贻贝、冻藏紫贻贝、自制紫贻贝干,贮藏自制紫贻贝干和市售紫贻贝干为研究对象,通过对紫贻贝表面附着微生物提取及高通量测序分析,获得不同紫贻贝样品中微生物组成及变化情况。实验结果表明,各样本中有效序列范围为30066～40563条、Operational taxonomic unit(OTU)范围为28～128,其中市售紫贻贝干OTU数最低,而冻藏紫贻贝OTU数相对较高;新鲜紫贻贝和自制紫贻贝干中主要是嗜冷杆菌属和不动杆菌属,冻藏紫贻贝干中主要为嗜冷杆菌属,贮藏紫贻贝干中主要为假单胞菌属和嗜甲基菌属,而市售紫贻贝干中优势菌群为葡萄球菌属和肠球菌属。不同状态下的紫贻贝样品的菌群组成均存在着较大距离,表明紫贻贝状态、加工过程及贮藏销售环境等,均对贻贝产品菌群组成产生较大的影响。研究结果可为保障紫贻贝制品的质量与安全提供参考。     

Determination of carbamate pesticides by a cholinesterase-based flow injection biosensor

A potentiometric flow injection-type biosensor developed in our laboratory was used for the determination of carbamate pesticides. The principle of the biosensor is based on the inhibition of enzyme by pesticides, which is dependent on the concentration of the pesticides present. The sensor system consisted of a reactor with acetylcholinesterase immobilized on controlled pore glass and a detector with tubular H⁺-selective membrane electrode. The sensor protocol established was applied to the analysis of nine carbamate pesticides. All the pesticides at 10⁻⁶ M inhibited the sensor enzyme, demonstrating the utility of the developed method. Treatment of the inhibited enzyme with pyridine-2-aldoxime restored nearly all of the enzyme activity allowing repeated use of the sensor. Calibration curves for carbaryl and carbofuran over the concentration range 10⁻¹¹–10⁻⁴ M were obtained. The lower detection limits were 10⁻¹⁰ M and 10⁻¹¹ M, respectively.     

Metabolic and biochemical responses of probiotic bacteria to oxygen

The interaction between oxygen and probiotic bacteria was studied by growing Lactobacillus acidophilus and Bifidobacterium spp. in 0, 5, 10, 15, and 21% oxygen in a hypoxic glove box. The metabolic responses of each probiotic strain in the different oxygen environments were monitored by measuring the levels of lactic acid and determining the lactate-to-acetate ratio. Biochemical changes induced by oxygen were examined by monitoring the specific activities of NADH oxidase, NADH peroxidase, and superoxide dismutase. In addition, the ability to decompose hydrogen peroxide and the sensitivity of each strain to hydrogen peroxide was also determined. With an increase in oxygen percentage, levels of lactic acid in L. acidophilus strains decreased, whereas the lactate-to-acetate ratio reduced in all the bifidobacteria tested. At 21% oxygen, the specific activities of NADH oxidase and NADH peroxidase, and the hydrogen peroxide decomposing ability of five probiotic strains was significantly higher than at 0% oxygen. The sensitivity of the probiotic strains to hydrogen peroxide however, remained unaffected in all the different oxygen percentages. Superoxide dismutase levels did not reveal any conclusive trend. In both L. acidophilus and Bifidobacterium spp., NADH oxidase and NADH peroxidase functioned optimally at pH 5. Growth in the various oxygen environments did not change this optimum pH.     

电化学生物传感器在农药残留检测中的应用研究进展

农药残留问题已对环境和人类健康造成严重威胁。因此,建立快速、灵敏、高效的农药残留检测方法势在必行。电化学生物传感器具有灵敏度高、选择性好、操作简便、成本低等诸多优点在分析领域有着广泛的应用。对农药残留检测中的样品前处理技术及电化学生物传感器应用的国内外最新进展进行了总结。     

高效液相色谱法快速测定烹炸油中苯并(a)芘的研究

苯并(a)芘是一种高活性间接致癌物,可通过食品加工等途径进入食品,本研究建立了高效液相色谱快速测定烹炸油中苯并(a)芘的方法.采用C18色谱柱分离;乙腈-水为流动相,流速1.2ml/min;荧光检测器检测,激发波长384nm,发射波长406nm;柱温30℃;进样量10μL.苯并(a)芘在1～ 40μg/L范围呈线性,线性回归方程为:Y=1.5758X +0.05705,相关系数为R2 =0.9998;方法检出限为0.1μg/kg,加标回收率大于95％,相对标准偏差(RSD)小于2.5％.该方法精确度和准确性好,样品预处理简单,分析时间短,适用于烹炸油中苯并(a)芘的定性定量要求,是烹炸油中苯并(a)芘含量的良好检测方法.     

Solar energy demand (SED) of commodity life cycles

The solar energy demand (SED) of the extraction of 232 atmospheric, biotic, fossil, land, metal, mineral, nuclear, and water resources was quantified and compared with other energy- and exergy-based indicators. SED represents the direct and indirect solar energy required by a product or service during its life cycle. SED scores were calculated for 3865 processes, as implemented in the Ecoinvent database, version 2.1. The results showed that nonrenewable resources, and in particular minerals, formed the dominant contribution to SED. This large share is due to the indirect solar energy required to produce these resource inputs. Compared with other energy- and exergy-based indicators, SED assigns higher impact factors to minerals and metals and smaller impact factors to fossil energetic resources, land use, and nuclear energy. The highest differences were observed for biobased and renewable energy generation processes, whose relative contribution of renewable resources such as water, biomass, and land occupation was much lower in SED than in energy- and exergy-based indicators.     

Photochemical removal of headspace oxygen by a singlet oxygen reaction

A new method is described for removing low levels of oxygen from enclosed atmospheres. The scavenging medium is a film of polymer such as ethyl cellulose or cellulose acetate which contains a sensitising dye and a singlet oxygen acceptor. The acceptor reacts with singlet oxygen formed by the sensitising action of the dye when illuminated. It is shown that the scavenging rate depends on the oxygen permeability of the polymer and on the dye concentration. Removal of oxygen to the detection limit of 0.03 % oxygen is achieved using erythrosin and difurfurylidinepentaerythritol in ethyl cellulose and in cellulose acetate. Potential applications include removal of oxygen from nitrogen-flushed packages of oxygen-sensitive foods, essences and chemicals, as well as from vessels for anaerobic microbiology.