Separation and fractionation of glutamic acid and histidine via origami isoelectric focusing.

We demonstrated the fractionation of two amino acids, glutamic acid and histidine, separated via isoelectric focusing (IEF) on filter paper folded and stacked in an origami fashion. Channels for electrophoresis were fabricated as circular zones acquired via wax printing onto the filter paper. An ampholyte solution with amphiphilic samples was deposited on all the circle zones, which was followed by folding to form the electrophoresis channels. IEF was achieved by applying an electrical potential between the anodic and cathodic chambers filled with phosphoric acid and sodium hydroxide solutions, respectively. A pH gradient was formed using either a wide-range ampholyte with a pH of 3 to 10 or a narrow-range version with a pH of 5 to 8, which was confirmed by adding pH indicators to each layer. The origami IEF was used to separate the amino acids, glutamic acid and histidine, by mixing with the ampholytes, which were deposited on the layers. The components in each layer were extracted with water and measured by high-performance liquid chromatography using pre-column derivatization with dansyl chloride. The results indicated that the focus for glutamic acid and that for histidine were at different layers, according to their isoelectric points. The origami isoelectric focusing achieved the fractionation of amino acids in less than 3 min using voltage as low as 30 V.

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady-state model to the current two-stage model of isoelectric focusing.

In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.

Electrophoretic mobilization in capillary isoelectric focusing by a weak acid or an acidic ampholyte as catholyte assessed by computer simulation.

Capillary isoelectric focusing (CIEF) with cationic electrophoretic mobilization induced via replacing the catholyte with the anolyte or a solution of another acid or amino acid was investigated by computer simulation for a wide range pH gradient bracketed between two amphoteric spacers and short electrode vials with a higher id than the capillary. Dynamic simulations provide insight into the complexity of the mobilizing process in a hitherto inaccessible way. The electrophoretic mobilizing process begins with the penetration of the mobilizing compound through the entire capillary, is followed by a gradual or steplike decrease of pH, and ends in an environment with a non-homogenous solution of the mobilizer. Analytes do not necessarily pass the point of detection in the order of decreasing pI values. Cationic mobilization encompasses an inherent zone dispersing and refocusing process toward the capillary end. This behavior is rather strong with phosphoric acid and citric acid, moderate with aspartic acid, glutamic acid (GLU), formic acid, and acetic acid and less pronounced in the absence of the cathodic spacer. The data reveal that optical detectors should not be placed before 90% of capillary length. Aspartic acid, GLU, formic acid, and acetic acid provide an environment with a continuously decreasing pH that explains their successful use in optimized two-step CIEF protocols.

Paper-based sample processing for the fast and direct MS analysis of multiple analytes from serum samples.

Amino acids are closely related to human health, and their rapid determination is important for the rapid diagnosis, timely treatment, and assessment of serious diseases. In this work, we propose a novel paper-based sample-processing device combined with isotope-dilution MS for the fast analysis of 11 amino acids from blood samples. By using an isoelectric focusing electrokinetic separation method, without the aid of carrier ampholytes and the addition of inhibitors, this approach uses only the characteristic of the isoelectric point of the target amino acids to achieve separation and purification from other coexisting components in the medium; it can meet the requirements for mass spectrometry detection. Driven by a DC voltage, a stable and sharp pH gradient (pH 3-10.5 over 5 mm) can be established in a glass-fiber paper-based fluidic channel with a MS-friendly electrolyte. Amphoteric species can be well separated from the complex blood matrix and concentrated into a narrow band in the channel within 2 min, which is 20 times faster than a commercial kit method. The method can be applied to both liquid and dry spot samples, and the cleaned sample band can be simply dissolved for direct IDMS detection in ESI MRM mode. This method is a promising strategy for the rapid MS-based detection of amino acids from serum without pre-separation via liquid chromatography.

CE determination of the thermodynamic pKa values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation.

Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pKa values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pKa values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pKa and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download).

Cytosolic delivery of quantum dots mediated by freezing and hydrophobic polyampholytes in RAW 264.7 cells.

Quantum dots (QDs) can be delivered efficiently inside macrophages using a freeze-concentration approach. In this study, we introduced a new, facile, high concentration-based freezing technology of low toxicity. We also developed QD-conjugated new hydrophobic polyampholytes using poly-l-lysine (PLL), a naturally derived polymer, which showed sustained biocompatibility, stability over one week, and enhanced intracellular delivery. When freeze-concentration was applied, the QD-encapsulated hydrophobic polyampholytes showed a higher tendency to adsorb onto the cell membrane than the non-frozen molecules. Interestingly, we observed that the efficacy of adsorption of QDs on RAW 264.7 macrophages was higher than that on fibroblasts. Furthermore, the intracellular delivery of QDs using hydrophobic polyampholytes was higher than those of PLL and QDs. In vitro studies revealed the efficient endosomal escape of QDs in the presence of hydrophobic polyampholytes and freeze-concentration. Collectively, these observations indicated that the promising combination of freeze-concentration and hydrophobic polyampholytes may act as an effective and versatile strategy for the intracellular delivery of QDs, which can be used for biological diagnosis and therapeutic applications.

Design of experiments as a valuable tool for biopharmaceutical analysis with (imaged) capillary isoelectric focusing.

Capillary isoelectric focusing is indispensable for characterizing charge heterogeneity and isoelectric points of biopharmaceuticals. However, there are many influencing parameters and therefore method development is challenging. This study was performed to obtain an in-depth understanding of the imaged CIEF methodology by applying a design of experiments approach. To describe the parameter's effects as objectively as possible, a polynomial regression model was derived for the most important responses. For this purpose, the reference monoclonal antibody suggested by the National Institute of Standards and Technology (NISTmAb) was used as test molecule. The total concentration and the mixing ratio of two types of carrier ampholytes and the added amounts of urea and l-arginine were selected as factors. The effects of these factors on 13 different responses such as resolution or pI values were investigated. In order to reduce the total number of experiments, a d-optimal design with 20 different parameter combinations and six replicates each was chosen. The most significant effects of the four factors were shown for the parameters related to separation efficiency and peak position. In addition, the extent of the factor's effect could be assessed. Depending on the selected factor combination, the pI value can differ up to approximately 0.15 pI units and the resolution value between main peak and adjacent basic peak can range from approximately 1.6 to 2.5, for example.

Impacts of cross-linker chain length on the physical properties of polyampholyte hydrogels.

Polymeric tissue engineering scaffolds have shown promise to aid in regeneration and repair of damaged tissue. In particular, nonfouling polymers have been proposed for eliminating biomaterial-induced concerns such as infection, scarring, and rejection by the immune system. Polyampholyte polymers are one class of nonfouling polymers that are composed of an equimolar mixture of positively and negatively charged monomer subunits. They possess nonfouling properties, bioactive molecule conjugation capabilities, and tunable mechanical properties. In this study, the influence of the cross-linker species on the degradation behavior, mechanical strength, and nonfouling properties of polyampholytes composed of a 1:1 molar ratio of [2-(acryloyloxy)ethyl] trimethylammonium chloride (positively charged) and 2-carboxyethyl acrylate (negatively charged) monomers was investigated. Specifically, the impact of ethylene glycol repeat units on the overall material performance was evaluated by synthesizing and characterizing hydrogels containing di-, tri-, and tetra-ethylene glycol dimethacrylate cross-linker species. The degradation studies were conducted for over 100 days in Sorenson's buffer with pH values of 4.5, 7.4, and 9.0 by tracking the swelling behavior and weight change over time. The mechanical properties were assessed using compression and tensile testing to failure. The retention of the nonfouling and protein conjugation capabilities was demonstrated using fluorescently labeled bovine serum albumin. The results demonstrate the tunability of both degradation behavior and mechanical properties through the cross-linker selection, without impacting the underlying nonfouling and biomolecule delivery capabilities. Therefore, it is concluded that polyampholyte hydrogels represent a promising platform for tissue engineering.

Carrier ampholyte-free free-flow isoelectric focusing for separation of protein.

Free-flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home-made carrier ampholyte-free FFIEF system was constructed via orientated migration of H+ and OH- provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R2 = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte-free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI-TOF-MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris-acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.

Molecular design of self-coacervation phenomena in block polyampholytes.

Coacervation is a common phenomenon in natural polymers and has been applied to synthetic materials systems for coatings, adhesives, and encapsulants. Single-component coacervates are formed when block polyampholytes exhibit self-coacervation, phase separating into a dense liquid coacervate phase rich in the polyampholyte coexisting with a dilute supernatant phase, a process implicated in the liquid-liquid phase separation of intrinsically disordered proteins. Using fully fluctuating field-theoretic simulations using complex Langevin sampling and complementary molecular-dynamics simulations, we develop molecular design principles to connect the sequenced charge pattern of a polyampholyte with its self-coacervation behavior in solution. In particular, the lengthscale of charged blocks and number of connections between oppositely charged blocks are shown to have a dramatic effect on the tendency to phase separate and on the accessible chain conformations. The field and particle-based simulation results are compared with analytical predictions from the random phase approximation (RPA) and postulated scaling relationships. The qualitative trends are mostly captured by the RPA, but the approximation fails catastrophically at low concentration.

Translocation of polyampholytes and intrinsically disordered proteins⋆.

Polyampholytes are polymers carrying electrical charges of both signs along their backbone. We consider synthetic polyampholytes with a quenched random charge sequence and intrinsically disordered proteins, which have a well-defined charge sequence and behave like polyampholytes in the denaturated state. We study their translocation driven by an electric field through a pore. The role of disorder along the charge sequence of synthetic polyampholytes is analyzed. We show how disorder slows down the translocation dynamics. For intrinsically disordered proteins, the translocation vs. rejection rates by the pore depends on which end is engaged in the translocation channel. We discuss the rejection time, the blockade time distributions and the translocation speed for the charge sequence of two specific intrinsically disordered proteins differing in length and structure.

Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.

A Review of the Environmental Degradation, Ecotoxicity, and Bioaccumulation Potential of the Low Molecular Weight Polyether Polyol Substances.

"Polyalkylene glycol" is the name given to a broad class of synthetic organic chemicals which are produced by polymerization of one or more alkylene oxide (epoxide) monomers, such as ethylene oxide (EO) and propylene oxide (PO), with various initiator substances which possess amine or alcohol groups. A generalization of this polymerization reaction is illustrated in Fig. 1.

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient.

Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300 µm, utilize ∼0.9 ng of protein with voltages below 10 V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300 µm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10 min yielding bands < 50 µm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20 µL for slab gel IEF to 0.002 µL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.

Modeling of formation and prevention of a pure water zone in capillary isoelectric focusing with narrow pH range carrier ampholytes.

This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling. Characteristics of two pH unit gradients with end components having pI values at or around 7.00 and conditions that lead to the formation of a water zone at neutrality were investigated. Data obtained reveal that a zone of water is formed in focusing with carrier ampholytes when the applied pH range does not cover the neutral region, ends at pH 7.00 or begins at pH 7.00. The presence of additional amphoteric components that cover the neutrality region prevent water zone formation under current flow. This situation is met in experiments with narrow pH gradients that end or begin around neutrality. Simulation data reveal that no water zone evolves when atmospheric carbon dioxide dissolved in the catholyte causes the migration of carbonic acid (in the form of carbonate and/or hydrogen carbonate ions) from the catholyte through the focusing structure. An electrolyte change in the electrode solution does not have an impact on the focusing part but does change the isotachophoretic pattern migrating behind the leading ion.

Steady-state protein focusing in carrier ampholyte based isoelectric focusing: Part I-Analytical solution.

The determination of an analytical solution to find the steady-state protein concentration distribution in IEF is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady-state protein distribution in carrier ampholyte based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I, and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field.

Steady-state protein focusing in carrier ampholyte-based isoelectric focusing: Part II-validation and case studies.

In this study, we systematically investigate the validity and applicability of an analytical model developed for carrier ampholyte-based isoelectric focusing (IEF). Three different IEF cases are considered in order to evaluate the efficacy of the approximate analytical results by comparison with high-resolution computer simulations. In the first case, three proteins are separated in a narrow pH range (6-9) by using 50 carrier ampholytes. In the second and third cases, the separation of proteins is studied in broad pH range (3-10) IEF by using 100 carrier ampholytes. Results obtained from the approximate analytical models are in very good agreement with the numerical results for IEF separation of cardiac troponin I, albumin, and hemoglobin in both narrow and broad pH ranges. The sensitivity of the analytical model is also tested for different initial mass ratios of proteins to ampholytes. No appreciable differences are observed between the approximate analytical and numerical results within the mass ratio range studied. The effect of a nominal electric field and/or a nominal pH gradient on protein focusing is also examined to demonstrate the effectiveness of the analytical model. Our results indicate that the use of both nominal electric field and pH gradient will result in erroneous peak concentrations for proteins. Finally, we describe the limitations of the approximate analytical solutions.

Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants.

One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1-10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3-10 with Pharmalyte 5-6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5-6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.

Application of an ampholine-functionalized hybrid organic-inorganic silica material for the SPE of aromatic amines.

An SPE cartridge based on an ampholine-functionalized hybrid organic-inorganic silica sorbent has been adopted for the analysis of aromatic amines including 4-aminobiphenyl, benzidine, 2-naphthylamine, p-chloroaniline, 2,4,5-trimethylaniline, and 3,3'-dichlorobenzidine. Crucial variables governing the extraction efficiency of the material such as the pH of sample, sample loading volume, solvent used for elution, and elution volume have been thoroughly optimized. The adsorption capacities for the six aromatic amines ranged from 0.17 to 1.82 µg/mg. The recoveries of aromatic amines spiked in textile samples ranged from 78.9 to 103.0%, with RSDs of 1.1-11.9% (n = 3). Moreover, the extraction efficiency of the ampholine-functionalized hybrid organic-inorganic silica sorbent was at least comparable with that of Oasis WCX.

The pH-sensitive polyampholyte nanogels: inclusion of carbon nanotubes for improved drug loading.

We report a simple and facile method to prepare a novel pH sensitive polyampholyte nanogel by copolymerizing vinylimidazole (VIM) with acrylic acid (AA) using functionalized single-walled carbon nanotubes (f-SWCNTs) (as reinforcing material) and cyanuric chloride via an intermolecular quaternization reaction. The polyampholyte nanogels have been characterized by various microscopic and spectroscopic methods. These studies reveal that incorporation of nanotubes in cross-linked copolymer of poly(vinylimidazole-co-acrylic acid) (PVI-co-AA) form polyampholyte nanogel with enhanced physical properties. The thermal experiments show that the introduction of f-SWCNTs into PVI-co-AA has significant impact on the thermal stability of nanogels. The rheological study showed that the nanogel is more viscoelastic than native gel. MTT assay indicates that the prepared polyampholyte gels possess biocompatibility and cell viability. The nanogel is also useful material to load water-soluble drug such as promethazine hydrochloride. The releasing profile of the drug from the nanogel clearly shows the pH-sensitive property of the material.