Characterization of artificial liposomes with free-flow electrophoresis

Free flow electrophoresis was used to examine the influence of active substances, lipid composition and preparation method on the surface charge of the liposomes. It is also possible to test the homogeneity of a liposome population with FFE.     

Conditions for purification of proteins by free-flow zone electrophoresis

The separation of proteins by free-flow zone electrophoresis is generally impaired by a number of secondary effects which cause spreading of the protein streams with resultant loss in resolution. A strategy is outlined, based on experimental observations and numerical modeling, which allows the operating conditions and separation chamber dimensions to be chosen so as to obtain complete separation of two proteins of known mobility. This approach takes into account some dispersive phenomena such as molecular diffusion, electroosmosis and residence time gradients. In some cases, the right operating conditions cannot be achieved on earth and electrophoresis separations in microgravity may then be justified.     

An online fiber-optic UV-visible detector for continuous free-flow electrophoresis

A PC-controlled, scanning online UV detector for continuous free-flow electrophoresis (CFFE) was designed to allow for single UV wavelength monitoring across a 1-D array of 48 longitudinal flow cells interfaced to a CFFE apparatus. In the detection scheme, the UV light is sequentially passed through each of the flow cells. The design integrates online acquisition of absorbance spectra of components separated by CFFE. Benzoic acid standard solutions were used to examine the performance of the detector. Chloroquine diphosphate and 4-nitrophenol were used as test solutes to determine the detector's ability to distinguish analytes separated in the CFFE.     

Do-it-yourself microelectrophoresis chips with integrated sample recovery

We present a microelectrophoresis chip that is simple to fabricate using the microfluidic tectonics (microFT) platform (Beebe, D. J. et al., Proc. Natl. Acad. Sci. USA 2000, 97, 13488-13493; Agarwal, A. K. et al.,. J. Micromech. Microeng. 2006, 16, 332-340). The device contains a removable capillary insert (RCI) for easy sample collection after separation (Atencia, J. et al.,. Lab Chip 2006, DOI: 10. 1039/b514068d). Device construction is accomplished in less than 20 min without specialized equipment traditionally associated with microelectrophoresis chip construction. microFT was used to build a PAGE device utilizing two orthogonal microchannels. One channel performs standard separations, while the second channel serves as an access point to remove bands of interest from the chip via the RCI. The RCI contains an integrated electrode that facilitates the removal of bands using electrokinetic techniques. The device was characterized using prestained proteins (Pierce BlueRanger and TriChromRanger). Samples were loaded into the microelectrophoresis device via a standard micropipette. An electrical field of 40 V/cm was used to separate and collect the proteins. The microPAGE device is simple to fabricate, benefits from microscale analysis, and includes an on-chip collection scheme that interfaces the macroworld with the microworld.     

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).     

多通道非接触电导检测装置用于自由流电泳分离在线检测

现有自由流电泳(FFE)装置因不具备在线检测功能,其实用性仍然存在明显不足.针对这一问题,该工作发展了一种多通道电容耦合式非接触电导检测(MC-C4 D)装置并开发了自动测量软件.MC-C4 D装置采用了并行分时的非接触电导检测技术,即由多个同样的非接触电导检测模块并行排列,而单个电导检测模块又由多个非接触电导检测池组...     

Single bubble in-tube microextraction coupled with capillary electrophoresis

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1 mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5 min SB-ITME yielded the enrichment factor values similar to those of 10 min HS-ITME. When 5 min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214 nm were 5.6–8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.     

Up-scaling capillary zone electrophoresis separations of polydisperse anionic polyelectrolytes with preparative free-flow electrophoresis exemplified with a soil fulvic acid

A scale-up of analytical capillary zone electrophoresis (CZE) to preparative free-flow electrophoresis (FFE) is described. FFE allows fractionations based on charge densities in larger amounts than in CZE, enabling further off-line analysis of the fractions. Model compounds (carboxylic acids and polystyrene sulfonates) showed a similar behavior in FFE as in CZE. Diffusion and electrodynamic distortion effects are more pronounced in FFE than in CZE. A soil fulvic acid was analyzed by CZE and fractionated by FFE. A comparison of the FFE fractions with CZE measurements of the same sample using the effective mobility scale showed good agreement of the two methods.     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

Combination of flow injection with electrophoresis using capillaries and chips

The technique of combined flow injection CE (FI-CE) integrates the essential favorable merits of FI and CE and can significantly expand the application of CE by utilizing the various on-line sample pretreatments and preconcentration of FI. The basic principles, instrumental developments, and applications of the FI-CE system from 2004 to 2006 are reviewed. The recent developments and applications of FI-CE are outlined.     

Reassemblable quasi-chip free-flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

An increasing number of small biosamples (e.g. proteins and enzymes) need micropreparation in lab. However, neither large-scale free-flow electrophoresis (LS-FFE) nor chip FFE (C-FFE) could fit the growing demands. Herein, a simple quasi-chip FFE (QC-FFE) was constructed. In contrast to C-FFE, the features of QC-FFE are as follows: (i) its separation chamber is reassemblable and rewashable avoiding discard of C-FFE due to blockage of solute precipitation in chamber; (ii) its chamber size is 45 mm × 30 mm × (80-500) μm (108-654 μL volume) having function of micropreparation; (iii) there are up to 16 outlets in QC-FFE bestowing fine fraction for micropurification. The QC-FFE was used for the micropurification of model enzyme of self-digestible trypsin in crude pancreatin. Under the given conditions, the purification factor of enzyme was 11.7, the specific activity reached 6236 U/mg, the run time for 19 μL sample purification was 45 s and the throughput of trypsin was 3.34 mg/h, and the yield of pure trypsin was 55.2%. All of the results show the feasibility of enzyme micropreparation via QC-FFE. The developed device and procedure have potential use to other micropurification of protein or peptide sample.     

On the surface modification of microchannels for microcapillary electrophoresis chips

This paper presents systematic investigation of the microchannel surface properties in microCE chips. Three popular materials for microCE chips, polydimethylsiloxane (PDMS), quartz, and glass, are used. The zeta potentials of these microchannels are calculated by measuring the EOF velocity to evaluate the surface properties after surface modification. The hydrophobic PDMS is usually plasma-treated for microCE applications. In this study, a new method using a high-throughput atmospheric plasma generator is adopted to treat the PDMS surface under atmospheric conditions. In this approach, the cost and time for surface treatment can be significantly reduced compared with the conventional vacuum plasma generator method. Experimental results indicate that new functional groups could be formed on the PDMS surface after treatment, resulting in a change in the surface property. The time-dependent surface property of the plasma-treated PDMS is then measured in terms of the zeta potential. Results show that the surface property will reach a stable condition after 1 h of plasma treatment. For glass CE chips, two new methods for changing the microchannel surface properties are developed. Instead of using complicated and time-consuming chemical silanization procedures for CE channel surface modification, two simple and reliable methods utilizing organic-based spin-on-glass and water-soluble acrylic resin are reported. The proposed method provides a fast batch process for controlling the surface properties of glass-based CE channels. The proposed methods are evaluated using PhiX-174 DNA maker separation. The experimental data show that the surface property is modified and separation efficiency greatly improved. In addition, the long-term stability of both coatings is verified in this study. The methods proposed in this study show potential as an excellent solution for glass-based microCE chip surface modification.     

PDMS extraction bars for the determination of volatile aromatic hydrocarbons in water and wastewater

In this study, the preparation and application of extraction bars of PDMS were investigated to preconcentrate and determine benzene, toluene, ethylbenzene, and xylene in water and wastewater by means of HPLC with fluorescence detection. Aliquot samples from hospital wastewater were used as the model effluent. The independent variables for the sorptive extraction were as follows: ionic strength (added amounts of NaCl); pH; temperature and time of absorption; temperature and time of desorption. Under optimized conditions, by using a factorial design, the suspended extraction bars could allow the determination of benzene, toluene, ethylbenzene, and xylene (1.20 ± 0.05 μg/L; 10.40 ± 0.02 μg/L; 1.80 ± 0.04 μg/L; 15.9 ± 0.04 μg/L, respectively) in hospital effluent (fortified samples), by recoveries of 71.9 ± 4.9 to 74.8 ± 5.6%. This procedure represents an innovation that eliminates the time‐consuming stage of vacuum microfiltration, and allows the determination of volatile organic compounds by HPLC. As far as we know, this procedure is original and represents an important contribution to the field.     

The combination of flow injection with electrophoresis using capillaries and chips

The combined flow injection capillary electrophoresis (FI-CE) system that integrates the essential favorable merits of FI and CE, can significantly expand the application scope of CE by exploring the various on-line sample pretreatments and preconcentration of FI. The principle behind this technique, some innovative designs of the split-flow interface, as well as novel applications to a variety of analytical problems, are reviewed and discussed. Some salient features and unique advantages of this technique are outlined.     

Development and optimization of an integrated PDMS based‐microdialysis microchip electrophoresis device with on‐chip derivatization for continuous monitoring of primary amines

An all‐PDMS on‐line microdialysis‐microchip electrophoresis with on‐chip derivatization and electrophoretic separation for near real‐time monitoring of primary amine‐containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene‐2,3,‐dicarboxyaldehyde/cyanide (NDA/CN^?), and the separation was performed using a 15‐cm serpentine channel. The analytes were detected using LIF detection.     

Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates

A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.     

Monolithically integrated dye-doped PDMS long-pass filters for disposable on-chip fluorescence detection

We report the fabrication of high quality monolithically integrated optical long-pass filters, for use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules directly into the microfluidic chip substrate, thereby providing a fully integrated solution that removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with 1200 microg mL(-1) Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4), >80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare favourably with commercially available Schott-glass long-pass filters, indicating that high quality optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The filters were found to be robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of low cost high quality integrated filters represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.     

Enhancing separation of histidine from amino acids via free-flow affinity electrophoresis with gravity-induced uniform hydrodynamic flow

In this paper, a novel mode of free-flow affinity electrophoresis (FFAE) was developed to indirectly enhance the separation of free-flow electrophoresis (FFE). In the mode of FFAE, a Ni(II) with high electric charge density and histidine (His) is chosen as a model ligand and target solute, respectively. Through the controlling of experimental conditions (10 mM pH 6.0 Na(2)HPO(4)-NaH(2)PO(4) with 2.0 mM NiCl(2)·6H(2)O background buffer), Ni(II) can combine with His and the combination leads to the high electric charge density of affinity complex of His-Ni(II) in contrast to the low density of free His molecule. But the ligand has weak interaction with uninterested amino acids. Thus, the mobility of His existing as His-Ni(II) is greatly increased from 14.5×10(-8) m(2) V(-1) s(-1) to 30.2 × 10(-8) m(2) V(-1) s(-1), while those mobilities of uninterested amino acids are almost constant. By virtue of the mode, we developed the FFAE procedure and conducted the relevant experiments. The experiments demonstrated the following merits of the FFAE technique: (i) clear enhancement of separation between the target solute of His and uninterested amino acids; (ii) simplicity, and (iii) low cost. Furthermore, the technique was used for the continuous separation of His from its complex sample, and the purity of His was near to 100%. All of the results demonstrate the feasibility of affinity separation in FFE. The developed FFAE may be used in the separation and pretreatment of some biological molecules (e.g. peptides).