Separation and characterization of gold nanoparticle mixtures by flow-field-flow fractionation

We show that using asymmetric flow-field-flow fractionation and UV-vis detector it is possible to separate, characterize, and quantify the correct number size distribution of gold nanoparticle (AuNP) mixtures of various sizes in the 5-60 nm range for which simple dynamic light scattering measurements give misleading information. The size of the collected nanoparticles fractions can be determined both in solution and in the solid state, and their surface chemistry characterized by NMR. This method will find widespread applications both in the process of "size purification" after the synthesis of AuNP and in the identification and characterization of gold-based nanomaterials in consumer products.     

Aggregation and dispersion of silver nanoparticles in exposure media for aquatic toxicity tests

Silver nanoparticles (AgNPs) are currently being very widely used in industry, mainly because of their anti-bacterial properties, with applications in many areas. Once released into the environment, the mobility, bioavailability, and toxicity of AgNPs in any ecosystem are dominated by colloidal stability. There have been studies on the stability or the aggregation of various nanoparticles (NPs) under a range of environmental conditions, but there is little information on fully characterised AgNPs in media used in (eco)toxicity studies. In this study, monodisperse 7, 10 and 20 nm citrate-stabilised AgNPs were synthesised, characterised and then fractionated and sized by flow field-flow fractionation (FFF) and measured with dynamic light scattering (DLS) in different dilutions of the media recommended by OECD for Daphnia magna (water flea) toxicity testing. Stability of NPs was assessed over 24 h, and less so over 21 days, similar time periods to the OECD acute and chronic toxicity tests for D. magna. All particles aggregated quickly in the media with high ionic strength (media1), resulting in a loss of colour from the solution. The size of particles could be measured by DLS in most cases after 24h, although a fractogram by FFF could not be obtained due to aggregation and polydispersity of the sample. After diluting the media by a factor of 2, 5 or 10, aggregation was reduced, although the smallest NPs were unstable under all media conditions. Media diluted up to 10-fold in the absence of AgNPs did not induce any loss of mobility or fecundity in D. magna. These results confirm that standard OECD media causes aggregation of AgNPs, which result in changes in organism exposure levels and the nature of the exposed particles compared to exposure to fully dispersed particles. Setting aside questions of dose metrics, significant and substantial reduction in concentration over exposure period suggests that literature data are in the main improperly interpreted and nanoparticles are likely to have far greater biological effects than suggested thus far by poorly controlled exposures. We recommend that the standard OECD media is diluted by a factor of ca. 10 for use with these NPs and this test media, which reduces AgNP aggregation without affecting the viability of the text organism.     

Gundelia tournefortii: Fractionation,Chemical Composition and GLUT4 Translocation Enhancement in Muscle Cell Line

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 μg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography–mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.     

A novel approach to improve operation and performance in flow field-flow fractionation

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate.     

Tandem hollow-fiber flow field-flow fractionation

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem HF5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a "trap" constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses.     

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF is carried out in an open-channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly applied field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution.In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.     

Experimental study on the retention of silica particles in gravitational field-flow fractionation effects of the mobile phase composition

Effects of mobile phase composition can play an effective role in modulating the retention of particles in gravitational field-flow fractionation (GFFF), the simplest and cheapest among field-flow fractionation (FFF) techniques. In the framework of an optimized procedure for the GFFF characterization of particulate systems, an experimental approach to the effects of the mobile phase composition on the retention of silica particles retention is presented. The role of the ionic strength and the presence of surfactant are emphasized, with special regards to the shape of the particles. Moreover, the first experimental evidence of potential-barrier GFFF is reported.     

Molecular fractionation in isotactic polypropylene during isothermal crystallization

The nucleation frequency of isotactic polypropylene shows for certain molecular weight distributions during isothermal crystallization a “stepwise” development of the nuclei as a function of time. The resulting curve can be fitted by a superposition of exponential functions assuming a separate nucleation density for each nucleation step. The multimodal nucleation is discussed as a consequence of molecular fractionation effects during crystallization. Received: 23 October 1997 Accepted: 12 May 1998     

蛙虹彩病毒cop基因的克隆表达及亚细胞定位

从蛙虹彩病毒(Rana gryliovirus,RGV)基因组中克隆了含凋亡相关结构域的新基因-cop(Caspaserecruit ment domain only protein,COP)基因的全部编码区,成功构建了重组表达载体,进行了原核表达,并在鲤鱼上皮瘤细胞(Epithelioma papulosumcyprini,EPC)中进行了亚细胞定位.序列分析表明,RGVcop基因全长288 bp,编码一个长为95 aa,分子量为10.4×103的推定蛋白.二级结构预测表明其含有5个α螺旋.同源性比对分析显示,RGVcop与其他6株虹彩病毒及人类cop相关蛋白同源性最高为94%,最低为33%.重组原核表达质粒pET28a-COP转化大肠杆菌BL21后,经IPTG诱导表达,获得一条约14×103的融合蛋白.重组真核表达质粒pEGFPN3-COP转染EPC细胞,经DAPI染色后,在荧光显微镜下观察显示重组蛋白在整个细胞中均有分布.