A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products

Immunochromatographic assays (ICAs) are considered as a suitable diagnostic tool for the detection of mycotoxins. Mycotoxins and especially, ochratoxin A are analytes with more demanding sensitivity requirements. To enhance the sensitivity of current immunochromatographic assays for ochratoxin A (OTA), a novel sensitive ICA was developed in this study. In the assay, microspheres enclosing fluorescent europium (III) [Eu(III)] nanoparticles (EuNPs) were used as a label for OTA monoclonal antibody (OTA-mAb) conjugation. Accordingly, assay was called time-resolved fluorescent immunochromatographic assay (TRFICA). The test strip was composed of three parts: a sample pad, nitrocellulose membrane and an absorbent pad. As for detection, a proper concentration of conjugated microspheres was pipetted into the microtube and sample extract was added to it. Then the strip was inserted into the tube and the fluid flow along the strip. The TRFICA results were obtained in 8 min and read by a portable TRFICA strip reader. The established method allows quantitative determination of OTA with limit of detection as low as 1.0 μg kg⁻¹ in the samples. For validation, spiked samples including wheat, maize, soybean and rice were respectively assayed by TRFICA and a standard high performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD), and good agreement of results was obtained between two methods.     

Thermal markers arising from changes in the protein component of milk

Classification of heat load applied to milk requires the detection of parameters appropriately related to the intensity of the heat treatment. Current analytical methods based on heat-induced changes in the protein component of milk have been directed either to determine the amount of protein-derived products arised from heat treatments or to evaluate the extent of thermal denaturation of milk proteins. Lately, a new analytical strategy has been developed according to the occurrence of three major whey proteins, namely bovine serum albumin (BSA), beta-lactoglobulin (βlg) and alfa-lactalbumin (αla), normally soluble at pH 4.6 in raw milk, in the pH 4.6 insoluble protein fraction recovered from heat-treated milk. The results have shown that pH 4.6 insoluble BSA, βlg and αla, as detected by ELISA in milk, can be regarded as thermal markers suited for either dairy process control or regulation purposes.     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

吡唑羧酸类金属配合物的合成、晶体结构及其与牛血清白蛋白的相互作用

以吡唑-3-甲酸、吡唑-4-甲酸为配体,采用溶液法分别与Cu Cl_2·2H_2O、Zn(NO_3)_2·6H_2O反应,合成了两种金属配合物并通过IR、X-射线单晶衍射仪对其晶体结构进行了表征。利用紫外-可见吸收光谱和荧光光谱研究了它们与牛血清白蛋白(BSA)的相互作用。实验结果表明,配合物与BSA发生强烈的结合,这些研究为人类研究药物分子与BSA相互作用的机理提供有价值的信息。     

荧光光谱法研究姜黄素与牛血清白蛋白的相互作用

考察了姜黄素与牛血清白蛋白(BSA)的相互作用机制。利用荧光光谱考察了两者之间的荧光淬灭类型,计算结合数(n)和结合常数,结果显示姜黄素与BSA作用可导致BSA发生静态淬灭,采用Stern-Volmer拟合方程测得结合常数Ka=4.52×108L/mol,结合位点数n=1.62。姜黄素可与牛血清白蛋白发生结合,导致牛血清蛋白发生静态荧光淬灭。     

高效疏水电荷诱导色谱介质的基团修饰及其色谱行为的初步研究

以粒径10μm、孔径300球形硅胶为载体,以γ-缩水甘油醚氧丙基三甲氧基硅烷(KH-560)为中间偶联剂,将4-巯基吡啶键合至硅胶上,制备了高效疏水电荷诱导色谱介质。通过单因素实验,探讨了反应温度、反应时间、KH-560的浓度对活化密度的影响,结果表明,在反应温度为75℃,反应时间为10 h条件下,KH-560的利用率大,可获得较宽范围的活化密度,此外还考察了缓冲液pH、反应温度、反应时间、4-巯基吡啶浓度对偶联密度的影响。结果表明,在pH=7.5的缓冲液,反应温度40℃,反应时间6 h下,4-巯基吡啶利用率高,通过控制4-巯基吡啶的浓度,可获得较宽范围的配基密度。最后,于高效液相色谱条件下,通过等度洗脱,考察了流动相pH对牛血清蛋白和溶菌酶保留因子的影响。并通过梯度洗脱,分离了不同等电点的牛血清蛋白和溶菌酶,验证了该介质的高效疏水电荷诱导色谱行为。     

Atom transfer radical polymerization (ATRP): A versatile and forceful tool for functional membranes

The progress in atom transfer radical polymerization (ATRP) provides an effective means for the design and preparation of functional membranes. Polymeric membranes with different macromolecular architectures applied in fuel cells, including block and graft copolymers are conveniently prepared via ATRP. Moreover, ATRP has also been widely used to introduce functionality onto the membrane surface to enhance its use in specific applications, such as antifouling, stimuli-responsive, adsorption function and pervaporation. In this review, the recent design and synthesis of advanced functional membranes via the ATRP technique are discussed in detail and their especial advantages are highlighted by selected examples extract the principles for preparation or modification of membranes using the ATRP methodology.