L-asparaginase release from Escherichia coli cells with aqueous two-phase micellar systems

A method was proposed to release and separate L-asparaginase (EC 3.5.1.1) from Escherichia coli ATCC 11303 cells with aqueous two-phase micellar systems. The systems were composed of K2HPO4 and Triton X-100. The method combines enzyme release with enzyme purification. The influence of Triton X-100 concentration, K2HPO4 concentration, and pH on the release and partition of L-asparaginase was investigated. Experimental results showed that E. coli cells treated with 9.4% (w/v) K2HPO4 and 15% (w/v) Triton X-100 at 25 degrees C for 15-20 h released nearly 80% of the enzyme. Most of the released enzyme was partitioned to the bottom phase (phosphate-rich phase). The effects of Triton X-100 concentration, K2HPO4 concentration, and pH on cloud point were also studied. Electron micrography indicated that the chemical treatment altered the inner structure of E. coli cells significantly.     

Extraction of humic acids in two-phase aqueous polymeric systems

Extraction of humic acids isolated from various soils was studied in polyethylene glycol(NH₄)₂SO₄, polyethylene glycol-dextran, and polyethylene glycol-dextran sodium salt two-phase aqueous polymeric systems. It was shown that, in all systems, humic acids are extracted into the polyethylene glycolrich phase. It was studied how the composition of the extraction system, molecular weight of the polymer, and the sample nature affect the distribution ratios of humic acids.     

Membrane osmometry of aqueous micellar systems

Summary The number average micellar molecular weights,M _n , were determined with the application of membrane osmometry, for various nonionic and a cationic surfactant, in aqueous solutions. These results show that these micelles are monodisperse, as also reported by other investigators. The dependence ofM _n on temperature was also studied. It was found that both the nonionic and the cationic micelles showed an abrupt change in logM versus temperature plots. In the case of nonionics, the abrupt change was observed at approximately 30 °C lower than the cloud-point.Presented at the 25th Colloid-Meeting in Munich, October 13–15, 1971.     

Interactions of halophenols with aqueous micellar systems

The interaction of a series of halophenols with sodium dodecyl sulphate and hexadecyltrimethylammonium bromide micelles was studied by different techniques (high-performance liquid chromatography, apparent acidity constant, spectral shift). The contributions of the halogens to the free energy of transfer from water to micelles are calculted. Correlations between the free energy of transfer for water/micellar solutions and for the classical octanol/water system are good. The application of micellar systems in chromatography and acid-base titrations is verified.     

Dissociation of acids in aqueous micellar systems

Incorporation in charged micelles induces large pKa shifts for a number of acids of varying type. Analysis of the measurements in terms of simple electrostatic theory is reasonably satisfactory in view of uncertainties regarding the net charge on the micelle and the exact character of its surface. The behavior of the long-chain fatty acids, whose mode of incorporation in micelles is least uncertain, confirms the suggestion of others that the effective dielectric constant at the micellar surface is quite low.     

Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.     

Thermodynamic molar properties of some aqueous micellar systems

Densities and ultrasonic velocities are measured then the partial molar volumes and adiabatic compressibilities are calculated at various concentrations of aqueous solutions of sodium dodecyl sulphate , sodium cholate and sodium taurcholate. These three detergents are known to form aggregates called micelles at specific concentrations. These concentrations are given the name , critical micelle concentration (cmc). Due to the nonlinear behaviour of the thermodynamic partial molar parameters with detergent's concentration no quantitative conclusions could be drawn . However, a reasonable qualitative model is put forward to explain the results.     

Enhancing the lateral-flow immunoassay for viral detection using an aqueous two-phase micellar system

Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5?×?10⁸ plaque forming units (pfu)/mL to 5?×?10⁷ pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA.

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.     

Selective separation of protein and saccharides by ionic liquids aqueous two-phase systems

In the present work, it was found that aqueous solution of a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium dicyanamide ([C₄mim][N(CN)₂]), could be separated into an aqueous two-phase system (ATPS) by inorganic salts such as K₂HPO₄ and K₃PO₄. The top phase is IL-rich, while the bottom phase is phosphate-rich. It was shown that 82.7%–100% bovine serum albumin (BSA) could be enriched into the top phase and almost quantitative saccharides (arabinose, glucose, sucrose, raffinose or dextran) were preferentially extracted into the bottom phase in a single-step extraction by [C₄mim][N(CN)₂] + K₂HPO₄ ATPS. The extraction efficiency of BSA from the aqueous saccharide solutions was influenced by the molecular structure of saccharides. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were combined to investigate the microstructure of the IL-rich top phase and the possible mechanism for the selective separation. It is suggested that the formation of the IL aggregate and the IL aggregate-BSA complex plays a significant role in the separation of BSA from aqueous saccharide solutions. This is the first example for the selective separation by ILs-based ATPSs. It is expected that these findings would have potential applications in bio-analysis, separation, and IL recycle.     

Enhancing the lateral-flow immunoassay for detection of proteins using an aqueous two-phase micellar system

The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05?μg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays.     

Protein diffusion across the interface in aqueous two-phase systems

We present a detailed study of the diffusive transport of proteins across a fluid phase boundary within aqueous two-phase systems. The aim of the work is to investigate whether local effects at the phase boundary cause a retardation of the diffusive transport between the phases. Possible modifications of interfacial mass transfer could be due to protein adsorption at the phase boundary or local electric fields from electric double layers. Experiments with a microfluidic system have been performed in which protein diffusion (bovine serum albumin and ovalbumin) within a bilaminated configuration of two phases containing polyethylene glycol and dextran is analyzed. A one-dimensional model incorporating phase-specific diffusion constants and the difference in chemical potential between the phases has been formulated. A comparison of experimental and simulation data shows a good overall agreement and suggests that a potential local influence of the phase boundary on protein transport is insignificant for the systems under investigation.     

Acoustic field assisted demixing of aqueous two-phase systems

Acoustic field assisted demixing was employed to decrease the demixing time in aqueous two-phase systems (polyethylene glycol-maltodextrin and polyethylene glycol-potassium phosphate). Application of acoustic field has decreased the demixing time in polyethylene glycol-maltodextrin by around twofold and up to about 3.2-fold in polyethylene glycol-potassium phosphate systems. Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time. In the polyethylene glycol-maltodextrin system, phase demixing was found to depend greatly on which of the phases iscontinuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence controlling the phase demixing rate. In case of the polyethylene glycol-potassium phosphate system, droplet coalescence was found to play a critical role in phase demixing. Addition of NaCl increased the demixing time and presence of Escherichia coli cells did not seem to have any influence on phase demixing.     

Monodisperse hydrogel microspheres by forced droplet formation in aqueous two-phase systems

This paper presents a method to form micron-sized droplets in an aqueous two-phase system (ATPS) and to subsequently polymerize the droplets to produce hydrogel beads. Owing to the low interfacial tension in ATPS, droplets do not easily form spontaneously. We enforce the formation of drops by perturbing an otherwise stable jet that forms at the junction where the two aqueous streams meet. This is done by actuating a piezo-electric bending disc integrated in our device. The influence of forcing amplitude and frequency on jet breakup is described and related to the size of monodisperse droplets with a diameter in the range between 30 and 60 μm. Rapid on-chip polymerization of derivatized dextran inside the droplets created monodisperse hydrogel particles. This work shows how droplet-based microfluidics can be used in all-aqueous, surfactant-free, organic-solvent-free biocompatible two-phase environment.     

乙醇-硫酸铵双水相萃取镉-碘化钾-罗丹明B离子缔合物

研究了在(NH4)2SO4存在下,碘化钾-罗丹明B-乙醇体系萃取Cd(Ⅱ)的行为及最佳分相条件。实验表明,在pH 1～3时,乙醇-(NH4)2SO4双水相体系对[CdI4]2-络阴离子的萃取率只有35.5%;加入罗丹明B后,该体系能完全萃取镉-碘化钾-罗丹明B形成的离子缔合物,而干扰离子Zn2+、Fe3+、Co2+、Cu2+、Ni2+不被萃取,实现Cd2+与上述离子的分离。     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

Electrokinetic demixing of aqueous two-phase polymer/salt systems

Electrokinetic demixing of aqueous two-phase polymer/salt systems is demonstrated, resulting in significant enhancement in demixing rates by about 1-4-fold. The effect of field polarity, field strength, volume ratio, and phase composition on phase demixing has been studied. Further the influence of these parameters on phase demixing could be explained based on the hydrodynamic flow-electroosmotic flow (HEF) model.     

Electrophoretic transport of solutes in aqueous two-phase systems.

Electrophoretic transport of proteins across the interface between the phases of an aqueous polymer two-phase system can be greatly impeded in comparison with transport within the individual phase. This effect can be controlled by modifying the affinity of the protein for a phase by suitable manipulations of such variables as pH. The effect is not caused by differences in the electrophoretic velocity between the two phases, nor by large changes in pH at the interface. An analogy exists between this phenomenon and the related subject of diffusion of electrolytes across the phase interface.