Application Of Aqueous Two-phase Systems Research Articles

Recent advances in drug delivery applications of aqueous two-phase systems.

Aqueous two-phase systems (ATPSs) are liquid-liquid equilibria between two aqueous phases that usually contain over 70% water content each, which results in a nontoxic organic solvent-free environment for biological compounds and biomolecules. ATPSs have attracted significant interest in applications for formulating carriers (microparticles, nanoparticles, hydrogels, and polymersomes) which can be prepared using the spontaneous phase separation of ATPSs as a driving force, and loaded with a wide range of bioactive materials, including small molecule drugs, proteins, and cells, for delivery applications. This review provides a detailed analysis of various ATPSs, including strategies employed for particle formation, polymerization of droplets in ATPSs, phase-guided block copolymer assemblies, and stimulus-responsive carriers. Processes for loading various bioactive payloads are discussed, and applications of these systems for drug delivery are summarized and discussed.

Continuous flow aqueous two-phase extraction of betalains in millifluidic channel

Aqueous Two-Phase System (ATPS) has a proven ability for the selective partitioning and purification of bioactives. This work explores the application of ATPS for the continuous flow extraction of bio-active solute (betalains) from an actual crude extract i.e. red beetroot extract. ATPS composed of Polyethylene Glycol (PEG) 6000 and ammonium sulphate is used for the differential partitioning of betalains and sugars. The continuous flow experiments were performed in millichannel (ID = 1.2 mm) with T-junction. The effect of residence time, multiphase hydrodynamics, and flow-rate ratios on the extraction performance in the continuous flow is studied. The effectiveness of the continuous flow extraction is compared with batch mode extraction of betalains. The continuous flow extraction offers 72.01 % extraction and 89.27 % equilibrium extraction efficiency for betalains with a residence time of 3.38 min. In comparison, batch extraction offered lower performance, with 60.35 % extraction and 74.81 % equilibrium extraction efficiency in 5 min. The improved performance for the continuous flow extraction is attributed to the uniform slug flow in the millichannel. Inclusively, this work develops a continuous flow process for the intensification of extraction of bioactives from the crude extracts of natural sources.

Application of Aqueous Two-Phase Systems with Thermoseparating Polymers (EOPO) as a Method for Extractive Fermentation with Neochloris oleoabundans

Extractive fermentation is an in situ method for the production and recovery of biomolecules of interest. Aqueous two-phase systems (ATPS) allow the product to be recovered in one phase of the system, reducing unit operations in the bioprocess. Thermosensitive polymers such as EOPOs are an interesting alternative to be applied in ATPS. In this work, different EOPOs were tested in an extractive fermentation strategy with the green microalgae Neochloris oleoabundans to provide a basis for future implementations of these systems in microalgae bioprocesses. Extractive fermentations were carried out with two EOPOs of different molecular weights (3900 and 12,000 g/mol) at concentrations of 10% and 15% (w/v). The microalga was incubated axenically under two different sets of conditions for 21 and 45 days, respectively. Cell counts were performed, and cell growth curves were obtained. Additionally, a semi-continuous and batch extractive fermentation assay was performed. The extractive fermentation with EOPO showed lower cell growth and a longer adaptation time of the microalgae in the fermentation, and EPS production yields of up to 8–23 g/L were obtained. Extractive fermentation is an interesting method to be implemented in microalgae cultures; however, further conditions need to be explored to achieve an appropriate bioprocess.

Thermodynamic modelling of the partitioning of lysozyme in aqueous two-phase systems composed of polyethylene glycol and salt at different temperatures

Knowledge about the interactions during the protein transfer is necessary to optimize the application of aqueous two-phase systems (ATPS) in purification processes. Theoretical knowledge combined with modelling and simulation can help to select the systems with the best partition values. Therefore, this study aimed to determine new interaction parameters through the contribution groups of lysozyme, using UNIFAC (Universal Functional Activity Coefficient) modelling. First, the partition of lysozyme was carried out, and the polymer molar mass (4000 or 6000), and the effects of the type of salt (Na2SO4 or Na3C6H5O7), the overall composition of the system (3 mooring lines), and temperature T = (293.15, 298.15, 303.15 and 308.15) K were evaluated. The results showed that lysozyme had a preference for the bottom phase of the ATPS system, the increase in the overall composition led to a tendency of increasing the partition coefficient (Kp), and the systems made up of higher molar mass polymers exhibited a bottom Kp value. The results of the thermodynamic parameters showed that the transfer of lysozyme in the phases was governed by entropy contribution. New interaction parameters of the contribution groups of lysozyme in the system phases were estimated. The UNIFAC model showed low deviation values. Therefore, the present results allow estimating new partitioning data for proteins presenting the same contribution groups, which can reduce the number of experiments and optimize the partitioning process.

Aqueous Two-Phase Systems Based on Ionic Liquids and Deep Eutectic Solvents as a Tool for the Recovery of Non-Protein Bioactive Compounds—A Review

Aqueous two-phase systems (ATPS) based on ionic liquids (IL) and deep eutectic solvents (DES) are ecofriendly choices and can be used to selectively separate compounds of interest, such as bioactive compounds. Bioactive compounds are nutrients and nonnutrients of animal, plant, and microbial origin that benefit the human body in addition to their classic nutritional properties. They can also be used for technical purposes in food and as active components in the chemical and pharmaceutical industries. Because they are usually present in complex matrices and low concentrations, it is necessary to separate them in order to increase their availability and stability, and ATPS is a highlighted technique for this purpose. This review demonstrates the application of ATPS based on IL and DES as a tool for recovering nonprotein bioactive compounds, considering critical factors, results and the most recent advances in this field. In addition, the review emphasizes the perspectives for expanding the use of nonconventional ATPS in purification systems, which consider the use of molecular modelling to predict experimental conditions, the investigation of diverse compounds in phase-forming systems, the establishment of optimal operational parameters, and the verification of bioactivities after the purification process.

Application of Aqueous Two-Phase System to the Purification of Persimmon Polyphenol Oxidase

Polyphenol oxidases (PPOs), which have recently become very popular among many researchers, catalyze the oxidation of phenolic compounds. Polyphenol oxidases are generally found in plants. Among them, persimmon (Diospyros kaki L.) is known as a good source for polyphenol oxidases. In this study, the polyphenol oxidase enzyme was purified from persimmon fruit using aqueous two-phase (ATPS). The optimized system was composed of 18% (w/w) PEG4000, 7% (w/w) NaH2 P04 and 1% (w/w) NaCl (pH 8.5, 25°C and 5 g). The PPO enzyme was obtained from the system by 4.8-fold purification with 191% activity recovery. The molecular weight of the enzyme, 28 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In summary, it has been shown that the PPO enzyme can be obtained from persimmon in a short time and at low cost using a non-chromatographic method-ATPS.

Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges.

The advancement of recombinant virus-like particle-based vaccines has attracted global attention owing to substantially safety and high efficacy in provoking a protective immunity against various chronic and infectious diseases in humans and animals. A robust, low-cost, and scalability separation and purification technology is of utmost importance in the downstream processing of recombinant virus-like particles to produce affordable and safe vaccines. Being a relatively simple, environmentally friendly, and efficient biomolecules recovery approach, aqueous two-phase systems have received great attention from researchers worldwide. This review aims to highlight the challenges and outlook in addition to the current applications of aqueous two-phase systems in downstream processing of virus-like particles. The efforts will confidently reinforce scholars' knowledge and fill in the valuable research gap in the aspect of concerning recombinant virus-like particle-based vaccines development, particularly related to the virus-like particles downstream production processes.

Partition of Tea Saponin with a Novel Recyclable Thermo-pH Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPS) have the advantages of environmentally friendly, high mass transfer efficiency, and mild extraction conditions. However, it is difficult to recycle these polymers, which limits the large-scale application of ATPS. In this study, a novel recyclable ATPS was constructed with thermo-responsive polymer PN and pH-responsive polymer PADB4.78 for the partition of tea saponin. PN represents poly-(N-isopropylacrylamide), and PADB4.78 represents poly-(acrylic acid-dimethylamine ethyl methacrylate-butyl methacrylate), where 4.78 in the subscript indicate the isoelectric point of the polymer. The recoveries of PN and PADB4.78 were 95.36% and 93.48%, respectively, after two cycles. Meanwhile, the phase formation mechanism of ATPS was studied by surface tension and low-field nuclear magnetic resonance (LF-NMR). The effects of polymer concentration, pH, temperature, types and concentrations of salt were investigated on tea saponin partition. In the 1.5% (w/v) PN/3.5% (w/v) PADB4.78 ATPS, the optimal partition coefficient (K) of crude tea saponin were 0.15 in the presence of 1.5 mM KCl at pH 7.6 and 25 °C while the extraction recovery (ERb) reached 92.13%. The K and ERb of tea saponin from tea seeds were 0.12 and 94.50% with 7.5 mM LiBr at pH 8.0 and 25 °C, respectively.

Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses

Green fluorescent protein (GFP) is a useful biomolecule in biotechnology; however, its price makes its widespread application prohibitive. To overcome this issue, recently, the use of aqueous two-phase systems (ATPS) for GFP purification was proposed as an alternative platform to reduce processing costs. Aligned with this goal, this study performed bioprocess modelling coupled with economic analysis using the software Biosolve to evaluate the potential and commercial applicability of ATPS for GFP purification. This work analysed a collection of fourteen ATPS to discriminate through production costs while also incorporating the concept of product purity into the calculations. The two best systems (a PEG-based and an ionic liquid (IL)-based ATPS) were placed in a full bioprocess at different scale models (1 to 100 L) to elucidate the viability of applying ATPS at large scale. Although the results showed that the PEG-based ATPS exhibit the lowest costs (between USD 3.5x103.g−1 at 1 L and USD 0.33x103.g−1 at 100 L), for further developments, the inclusion of an ATPS granting a higher purity is desired for the development of simpler bioprocesses. Therefore, as a third approach in this work, a sensitivity analysis was performed to determine the impact of varying different model parameters (recovery yield, material costs discount and production titre), to elucidate the circumstances under which the IL-based system can overcome the production costs of the traditional PEG-based ATPS. The results indicate that the best cost-effectiveness approach is to improve the production titre (although it can affect all ATPS studied), as an increase from 1.33 to 3.8 g/L is enough for the IL-based ATPS to be less expensive than the traditional system at all analysed scales. This study demonstrates that ATPS can greatly reduce GFP manufacturing costs, which can potentially help to popularize new applications of fluorescent proteins that are currently mostly restricted to research kits due to their high prices.

Extraction of yttrium from fluorescent lamps employing multivariate optimization in aqueous two-phase systems

This paper presents for the first time the application of aqueous two-phase systems for yttrium recovery from fluorescent lamp residues, a rare earth element with high added value and widely used in the technology industry. A multivariate study was carried out, using experimental design, for yttrium extraction employing ATPS using alizarin red as extracting agent, in order to expand the possibilities of the ATPS application in industrial scales. The optimized variables, employing 26–1 fractional design, were pH (3.00–9.00), extracting agent concentration (2.3–23.0 mmol kg−1), tie-line length (TLL), ATPS-forming copolymers (L35 and L64) and electrolytes (Na2SO4 and C6H5Na3O7·2H2O), and mass ratio between top and bottom phases (mTP/mBP = 0.5–2.0). It was observed for the first time, that the metal ions partition in ATPS is mainly influenced by the salt-forming anion species distribution as a function of pH at high TLL values. The extraction of yttrium was optimized in a 23 factorial design with no lack of adjustment and it was able to predict the extraction response with accuracy close to 96%. The optimized conditions of yttrium extraction were applied using a leach liquor from real sample of fluorescent powder. After five extraction steps, 90% of yttrium was selectively recovered.

Application of aqueous two-phase systems for the extraction of pharmaceutical compounds from water samples

In this study, the proposal was to use aqueous two-phase systems (ATPS) as a sample preparation technique for the determination of five pharmaceuticals, classified as emerging contaminants (EC): ibuprofen, paracetamol, ciprofloxacin, norfloxacin and amoxicillin. The evaluation of this study was performed through the partition coefficient (K) of each pharmaceutical by varying some parameters of the system, such as the pH value of the medium, the nature of the ATPS-forming electrolyte and polymer, the tie-line length (TLL), the system temperature and the mass ratio between the top and bottom phases of the ATPS. Values of K between 9.30 and 1150 were obtained for the five pharmaceuticals during the system optimization. The ATPS also proved to be efficient as a sample preparation technique in the partition of the studied pharmaceuticals when applied to water samples, including drinking water, surface water and filtered water from a water treatment plant. The values of the partition coefficients found in these complex samples ranged from 2.49 to 74.2.

The temperature influence on the phase behavior of ionic liquid based aqueous two-phase systems and its extraction efficiency of 2-chlorophenol

With the increasing research on the aqueous two-phase systems (ATPS), the ATPS containing ionic liquid (IL) and salt have been widely used in the extraction and purification process. In this work, the partitioning of 2-chlorophenol in novel ATPS composed of n-butylpyridinium trifluoromethanesulfonate and salts (ammonium sulfate, sodium dihydrogen phosphate) is evaluated. Firstly, the cloud point titration method was used to determine the binodal curves data experimentally at four temperatures and atmospheric pressure. And Merchuk equation was used to fit the binodal curves. Then, the gravimetric method was used to initially determine the tie-lines data and the concentration of all the ions in the top and bottom phases were determined by ion chromatography method to study the ion exchange in the ATPS. The reliability of tie-lines was evaluated by Othmer-Tobias and Bancroft equations. Finally, the influence of types of salts and temperature on the phase diagrams were studied carefully. Besides, in order to analyze the application of ATPS in the extraction, the partition coefficient of 2-chlorophenol was calculated at the same total composition while at different temperature. The ATPS formed by n-butylpyridinium trifluoromethanesulfonate, ammonium sulfate, and water at 298.15 K was found to have the maximum amount of partition coefficient and extraction efficiency.

The Application of Aqueous Two-phase System in the Extraction of Natural Products from Chinese Herbal Medicine: A Review

Background: In the past decades, Chinese herbal medicine has attracted worldwide attention because they contain a variety of active ingredients which are beneficial to human health. As a result, there is a growing interest in the extraction of these substances. However, traditional extraction methods not only need a large amount of extractant, but are also time-consuming, moreover, the extraction efficiency is extremely poor and tedious purification steps are required to purify the crude extract. Thus, researchers hope to find an alternative method for the extraction of these components and the aqueous two-phase system (ATPS) seems to be one. Objective: This review focuses on introducing the properties of the aqueous two-phase system and summarizing the application of ATPS in the extraction of natural products. Meanwhile, this review also provided a guideline to researchers who wish to design a suitable ATPS for a specific target and how to amplify it to industrial-scale.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Phase Diagrams, Densities, and Refractive Indexes of Aqueous Two-Phase Systems Comprising (F68, L64, or PEO1500) + (Ammonium, Sodium, or Potassium Thiocyanate Salts) + Water: Effect of Cation and Type of Macromolecule

In extraction procedures, the more commonly used aqueous two-phase systems (ATPS) comprise mainly water, salt, and macromolecule, particularly the macromolecule poly(ethylene oxide) (PEO). However, one limitation of such ATPS is their capacity to separate compounds that are more hydrophobic. One possible solution to overcome this restriction is the use of ATPS formed with triblock copolymers, which are more hydrophobic and therefore enable the extraction of hydrophobic solutes. In addition, the range of applications of ATPS formed with thiocyanate salts can be broader, mainly to extract metal ions. In view of this, equilibrium data were acquired in this work by constructing phase diagrams for ATPS comprising macromolecules [poly(ethylene oxide), PEO, or (poly(ethylene oxide))-(poly(propylene oxide))-(poly(ethylene oxide)) triblock copolymers, F68 or L64] + thiocyanate salts (ammonium, sodium, or potassium) + water at 25.0 °C. The influence of the nature of the cation on the formation of the ATPS was inves...

Synthesis of pH-responsive polymers forming recyclable aqueous two-phase systems and application to the extraction of demeclocycline

The application of aqueous two-phase systems (ATPS) to bioseparation and biocatalyst engineering has attracted interest. However, despite the distinct advantages of the technique, the scaling up of ATPS is limited by the cost of the system components. In this study, two novel recyclable pH-responsive polymers were synthesized and tested for the partition of demeclocycline. The recoveries of two recyclable pH-responsive polymers could reach over 95.0% by only adjusting the solution pH to the isoelectric points of the polymers. The control variate method was used to investigate the effects of polymerization conditions on polymer synthesis. Polymer PADB4.6 was firstly synthesized in 500 l reactor with two-stage heating method (25–40 ℃ and 40–62 ℃), as the heating rate is an important factor in the scale-up process. The polymer characteristics (molecular weight, viscosity, surface tension, and zeta potential) were studied to understand their influences on the synthetic process. Furthermore, the phase formation mechanism was studied with low-field nuclear magnetic resonance (LF-NMR). The optimal partition coefficient and recovery of demeclocycline using the recyclable ATPS were 0.24 and 82.9% at pH 6.30 in presence of 20 mmol/L MgSO4.

Equilibrium data and thermodynamic studies of α-amylase partition in aqueous two-phase systems

Liquid-liquid equilibrium data (LLE) were obtained for aqueous two-phase systems (ATPS) composed of polyethylene glycol (average molar mass of 1500 g mol−1), sodium citrate/citric acid and water, at different pH (4.0, 5.0, 6.0 and 7.0) and temperatures T = (303.15 and 313.15) K. The thermodynamic study of the phase equilibrium was performed. The experimental results of the LLE were correlated using the UNIversal Functional Activity Coefficient (UNIFAC) model. The salting-out effect evaluated using the model of the effective excluded volume (EEV). Partitioning of the α-amylase (EC 3.2.1.1) was investigated as a function of pH, temperature, and tie line length (TLL). It was observed that the temperature had no influence on the formation of the biphasic region. Increments in pH value resulted in the raising of biphasic region. Isothermal titration calorimetry (ITC) assays was performed in order to deeply understanding the intermolecular interactions involved in the α-amylase partition and the driving forces that govern this process. It was measured the Gibbs free energy transfer (ΔtrG), transfer enthalpy variation (ΔtrH) and transfer entropy variation (ΔtrS) during the enzyme partition, as a function of the pH, temperature and TLL. For α-amylase partition, it was verified that at pH 4.0 the enzyme had a strong tendency to transfer to the polymer-rich phase. At pH 5.0 α-amylase showed affinity for the salt-rich bottom phase, while at pH 6.0, the partition coefficient of the enzyme increased as temperature was reduced to 303.15 K. The opposite behavior was noted for α-amylase at pH 7.0. Thermodynamic analysis based on isothermal titration microcalorimetry indicated the partition of α-amylase partition in the ATPS at pH 4.0 and 7.0 and 313.15 K was accompanied by endothermic heat and was entropically driven to the upper phase. Results of yield (95.373%) parameter indicated the applicability of ATPS for amylase purification.

Model of protein isoforms analysis by aqueous two-phase systems: Methodology importance in clinical biochemistry and biopharmaceutical production

Aqueous phase partitioning has a long history of applications to the analytical characterization of biomolecules. However, process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The application of aqueous two-phase systems (ATPSs) has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This type of separation and purification system has also been successfully used for the separation of virus and virus-like particles. The advantage of this technique is that it may be used to monitor the aforementioned changes for purified proteins as well as for proteins in biological fluids, and that it is readily adaptable to automated high-throughput screening. However, the wide use of this technology has been diminished by the lack of a clear understanding of the factors and mechanisms that govern the behavior of proteins in these systems. It has prevented the development of analytical models that assist the rational design of these systems. This work has revised the development of ATPSs (preparation and sampling techniques), and also highlighted the knowledge gap in the ATPS.

Enhanced enzymatic reaction by aqueous two-phase systems using parallel-laminar flow in a double Y-branched microfluidic device

Although there are several reports showing the employment of aqueous two-phase systems (ATPS) for biotransformation in large-scale, the industrial application of ATPS is still hampered by their drawbacks such as slow diffusive mass transfer, long settling time for phase separation, and batch processes. Here, we propose for the first time a continuous-flow process based on a suitably designed ATPS parallel-laminar flow in a double Y-branched microfluidic device using for enzymatic catalysis. In a model urease reaction, polyethylene glycol (PEG) 8 kDa and dextran (Dex) 500 kDa are selected as polymer solutions, in which the enzymes settle mainly in the Dex phase while the products partition mainly to the PEG phase during short residence time. On account of rapid micro-transport, as well as integrate one single step of biocatalysis and separation process, obvious intensification of enzymatic catalysis in such ATPS microfluidic platform is demonstrated. Compared with the conventional ATPS in a beaker under gentle stirring, the enzymatic reaction rate in ATPS microfluidic is 500 times higher. Furthermore, the effects of flow rates, substrate concentration, residence time, and recycling number on the reaction rate and conversion rate are evaluated, respectively. Our study provides an all-aqueous, micro-scale and coupled reaction-separation platform for the process intensification of enzymatic catalysis, which has not only theoretical significance, but also practical value for biotransformation-based fields, such as biomedical, pharmaceuticals and food applications.

Extraction separation of the components of leach liquors of batteries

Waste batteries contain toxic metals (Pb, Ni, Zn, Fe, etc.) in high concentrations. In order to prevent harmful effects on human health and environment, it is necessary to recycle them. In this paper, prospective applications of aqueous two-phase systems for isolating and separating the components of leach liquors of batteries have been considered. The extraction of metal cations in the systems based on poly(ethylene oxide) with molecular masses of 1500 and ammonium sulfate depending on the process conditions has been studied. A flow diagram of the separation of the components of leach liquors that uses a studied aqueous two-phase system in the presence of KSCN complexing agent has been suggested.