Polyacrylamide Membranes Research Articles

A cyclometalated N-heterocyclic carbene and acetylacetonate ligands in a phosphorescent Pt(II) dye for sensing glucose

New β-diketonate platinum (II) complexes, containing a cyclometalated N-heterocyclic carbene [Pt(Naph^C*iPr)(acac)] (3A) (HNaph^C*-κC* = 3-isopropyl-1-(naphthalen-2-yl)-1H-imidazole-2-ylidene), or a cyclometalated pyrazole, [Pt(Naph^Npz)(acac)] (3B) (HC^Npz = 1-(naphthalen-2-yl)-1H-pyrazole) and [Pt(Naph^Ndmpz)(acac)] (3B') (HC^Ndmpz = 1-(naphthalen-2-yl)-1H-3,5-dimethylpyrazole) have been prepared and characterized. Their absorption and emission properties in films of ethyl cellulose (EC) were determined along with those of the already reported for complex [Pt(Naph^C*Me)(acac)] (3A'). They showed that all four β-diketonate complexes display a bright phosphorescent emission with maxima in the blue region (λmax ∼ 480 nm for 3A and 3A'; 490 nm 3B and 3B'). The higher quantum yield (QY), longer decay times and greater oxygen sensitivity were exhibited by the Naph^C* derivatives, compared to the Naph^N ones. Polyacrylamide membranes with entrapped 3A' as dye, and glucose oxidase (GOx) enzyme were used for monitoring glucose level. The RSD is about 5% and the detection limit is at ∼5·10−4 M, with a response time usually of 10–15 min working in stop-flow mode. These platinum-based membranes respond reversibly to glucose for, at least, 20 measures. 3A’ is the first Pt(II) complex bearing a cyclometalated N-heterocyclic carbene ever used as dye for sensing glucose.

Heat and moisture management in membranes containing magnetic field-induced oriented nanosurfaces

ABSTRACTThis work investigates how the orientation of graphene oxide (GO) and reduced graphene oxide (rGO) affect the moisture and heat transfer of poly acryl amide membranes. In so doing, iron oxide (Fe3O4) nanoparticles were deposited onto synthesized GO and rGO using the solvothermal method. Magnetic nanosheets were drawn to the through–plane direction of the prepared membranes by applying a magnetic field for different time lengths on the solution during casting. Fourier transform infra-red spectroscopy, X-ray diffraction, transmission electron microscopy (SEM), water vapor permeability (WVP), and dynamic heat transfer simulation apparatus were performed to detect the structures and characteristics of the nanosheets and membranes. The SEM confirmed the orientation of nanosheets in the magnetic field. The oriented polyacrylamide (PAAm)/mrGO membranes showed a higher transient temperature and WVP compared to the membranes with oriented and random GO. With an increase in the duration of the application of the magnetic field, the results of the above-mentioned tests improve. The oriented PAAm/mrGO membranes can have applications in wound dressing and protective clothing.

Ethanol:water blends separation using ultrafiltration membranes of poly(acrylamide‐co‐acrylic acid) partial sodium salt and polyacrylamide

AbstractIn this work, poly(acrylamide‐co‐acrylic acid) partial sodium salt (P(AAM‐co‐AA)Na), and polyacrylamide (PAAM) membranes were synthesized for separation of ethanol:water blends. The membrane characteristics were evaluated to determine their ability to separate mixtures of ethanol:water and get dehydrate ethanol (95 %). Synthesized membranes showed higher selectivity to ethanol compared to existing polymeric membranes. The ethanol:water blends had an ethanol concentration of 0.10 to 0.70 L/L. The results showed that a mixture of ethanol:water (75:25 L/L) can be concentrated up to 95 % ethanol. The membranes’ stability were studied in absolute ethanol and ethanol:water blend (30:70 L/L) for a period of 30 days of permanent exposure. The membrane deterioration is due to the chemical modification of the membrane active surface layer. It was found that the membranes have a high selectivity (350 times higher) compared to existing membranes, and the substantial and flow of water was 12.15 kg · m−2 · h−1.

High-temperature proton exchange membranes from microporous polyacrylamide caged phosphoric acid

To enhance the H3PO4 loading and therefore proton conductivity, freeze-dried microporous polyacrylamide (PAM) was doped with a highly concentrated H3PO4 aqueous solution, leading to high percentages of cages of H3PO4 in the PAM membranes for potential use in high-temperature proton exchange membranes (PEMs). A H3PO4 fraction of 92.0 wt% and a proton conductivity as high as 0.132 S cm−1 at 183 °C were obtained. The H3PO4 loading by microporous PAM membranes obeys a Fickian diffusion process. Protons migrate along the channels from the H3PO4 bonded PAM framework or PO⋯H–O hydrogen bonds of H3PO4 molecules according to the Grotthuss mechanism. This new concept, in combination with high conductivity, good H3PO4 retention and simple-synthesis, opens a new approach to high-temperature PEMs.

Microchip electrophoresis of DNA following preconcentration at photopatterned gel membranes

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double-stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size-dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single-stranded oligonucleotide DNA (20-mer ssDNA). Baseline resolution of a six-band dye-labeled ladder with fragments 100-2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000-15,000 plates/cm, and detection limits as low as 1 pM per single dye-labeled fragment. The degree of preconcentration is tunable by at least 49-fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5-fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength.

Microscale Isoelectric Fractionation Using Photopolymerized Membranes

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. μIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.

Homogeneous Immunosubtraction Integrated with Sample Preparation Enabled by a Microfluidic Format

Immunosubtraction is a powerful and resource-intensive laboratory medicine assay that reports both protein mobility and binding specificity. To expedite and automate this electrophoretic assay, we report on advances to the electrophoretic immunosubtraction assay by introducing a homogeneous, not heterogeneous, format with integrated sample preparation. To accomplish homogeneous immunosubtraction, a step-decrease in separation matrix pore-size at the head of a polyacrylamide gel electrophoresis (PAGE) separation channel enables "subtraction" of target analyte when capture antibody is present (as the large immune-complex is excluded from PAGE), but no subtraction when capture antibody is absent. Inclusion of sample preparation functionality via small pore size polyacrylamide membranes is also key to automated operation (i.e., sample enrichment, fluorescence sample labeling, and mixing of sample with free capture antibody). Homogeneous sample preparation and assay operation allows on-the-fly, integrated subtraction of one to multiple protein targets and reuse of each device. Optimization of the assay is detailed which allowed for ~95% subtraction of target with 20% non-specific extraction of large species at the optimal antibody-antigen ratio, providing conditions needed for selective target identification. We demonstrate the assay on putative markers of injury and inflammation in cerebrospinal fluid (CSF), an emerging area of diagnostics research, by rapidly reporting protein mobility and binding specificity within the sample matrix. We simultaneously detect S100B and C-reactive protein, suspected biomarkers for traumatic brain injury (TBI), in ~2 min. Lastly, we demonstrate S100B detection (65 nM) in raw human CSF with an estimated lower limit of detection of 3.25 nM, within the clinically relevant concentration range for detecting TBI in CSF. Beyond the novel CSF assay introduced here, a fully automated immunosubtraction assay would impact a spectrum of routine but labor and time-intensive laboratory medicine assays.

Nanosieving for rapid, solution‐phase immunoassays

We demonstrate an innovative nanosieving technique for rapid solution-phase measurement of antibodies. Polyacrylamide membranes were fabricated by in situ UV lithography in glass microchannels. Membrane pore-size was optimized to exclude large species, while small proteins (<10 kDa) electrophoretically migrate through the membrane. For detection of unlabeled antibodies, buffer containing anti-insulin IgG was electrophoretically loaded into the microchannel. Owing to the large size of IgG, the antibody was retained at the membrane interface. A known concentration of FITC-labeled insulin (Ins*) was introduced. Retention of Ins* at the membrane indicated formation of Ins*-IgG complexes (see figure), as unbound Ins* migrates through the membrane when IgG is not present. Fluorescence signal at the membrane enabled IgG quantitation. The device exploits advantages of scale-dependent transport processes in confined geometries, namely for rapid immunorecognition. Unlike protein microarrays, no surface immobilization is necessary. The reported assay could allow study of previously unusable binding pairs and binding kinetics. Funding from UC Berkeley is gratefully acknowledged.

‘Gate effect’ in templated polyacrylamide membranes influences the electrotransport of proteins and finds applications in proteome analysis

Templating is an effective way for the structural modifications of a material and hence for altering its functional properties. Here protein imprinting was exploited to alter polymeric polyacrylamide (PAA) membranes. The sieving properties and selection abilities of the material formed were evaluated by studying the electrically driven transport of various proteins across templated PAA membranes. The sieving properties correlated with the templating process and depended on the quantity of template used during the polymerisation. For 1 mg/mL protein-templated membranes a 'gate effect' was shown, which induced a preferential migration of the template and of similar-size proteins. Such template preferential electrotransport was exploited for the selective removal of certain proteins in biological fluids prior to proteome analysis (depletion of albumin from human serum); the efficiency of the removal was demonstrated by analysing the serum proteome by two-dimensional electrophoresis experiments.

Effect of different electrolytes on the swelling properties of calyx[4]pyrrole-containing polyacrylamide membranes

Calix[4]pyrrole ( 1) was synthesized and characterized and this macrocycle was incorporated in polyacrylamide gels. The presence of meso-octamethyl-porphyrinogen inside of gel was checked using infrared spectroscopy, differential scanning calorimetry, and swelling studies. The swelling degree of these hydrogels in equilibrium with different electrolytes (NaCl, LiCl, KCl, CaCl 2 and AlCl 3) was measured in a concentration range 0.1–0.5 mol dm −3. Although no significant alterations in the swelling degree can be found for the different 1:1 electrolytes, when the cation charge of unsymmetrical electrolytes increases, the gel swells in a significant way. This swelling process is enhanced by the presence of calyx[4]pyrrole. The effect of alkaline hydrolysis of polaycrylamide-based hydrogels was also studied. The hydrolysed hydrogels collapse in the presence of the electrolytes; this behavior is dependent on the hydrolysis degree, electrolyte charge and calyx[4]pyrrole presence and concentration; the latter leads to polyacrylamide with tailor-made properties.

Luminescence-Based Optical Fiber Chemical Sensors

A scheme for the simultaneous determination of temperature and analyte concentration for application in luminescence-based chemical sensors is proposed. This scheme is applied to an optical oxygen sensor, which is based on the quenching of the fluorescence of a ruthenium complex. Temperature measurement is performed using the excitation radiation and an absorption long-pass filter. Preliminary results are presented that show the viability of an oxygen measurement that is independent of temperature and optical power level. The possibility of self-referenced temperature measurements with semiconductor nanoparticles is also investigated. In order to optimize the sensor design, several different optical fiber probe geometries for oxygen sensing are tested and compared, including different methods of coupling radiation into the optical fiber system. Polyvinyl alcohol (PVA) and polyacrylamide membranes are tested as supports for sensor immobilization in fiber-optical pH sensing devices in aqueous solution. Some results are presented that show the feasibility of using fiber-optical pH indicators for remote monitoring.

Evaluation of Solution Phase Isoelectrofocusing as Part of Proteomics Strategies

Solution isoelectrofocusing has been evaluated for prefractionation of nuclear proteins from human cancer MCF-7 cells. Two separation devices were exercised using commercial polyacrylamide membranes, which carry embedded immobiline mixtures to establish pH boundaries between chambers. Protein recovery was quantified and 2-D gel electrophoresis was used to evaluate resolution. Solution isoelectrofocusing provides satisfactory separations and decreases the complexity of the protein mixture, however rates of recovery need to be improved.

Protein transfer through polyacrylamide hydrogel membranes polymerized in lyotropic phases.

A way to control the average pore size in cross-linked polyacrylamide-based membranes is by altering the ratio of cross-linker to acylamide monomer. Larger pore sizes are prepared with a minimum amount of cross-linker, resulting in membranes that are mechanically weak and have short lifetimes. The aim of this study was to prepare cross-linked polyacrylamide membranes with large pore sizes and with good mechanical integrity. The methodology was to carry out the polymerization in a template, formed from the self-aggregation of surfactant. Two surfactant templates were used, and their pore size was examined with proteins of different sizes. The surfactants chosen for this study were sodium dodecyl sulfate (SDS, ionic surfactant) and TERIC BL8 (nonionic surfactant), both of which have very different aggregation properties. The data showed that at 10% and greater of TERIC BL8, a very different and open gel structure is formed, in which the pore size was significantly increased. SDS seemed to have little effect on the pore size. The data suggests that the gel structures for both surfactants up to 4% (w/v) are similar and micellular, because SDS is known to favor a micelle structure. Above 4% (w/v), TERIC BL8 then goes through a change in its lyotropic phase, thus, producing membranes of a large pore size. In conclusion, the pore size and gel structure of polyacrylamide hydrogel membranes can be significantly increased using TERIC BL8 (nonionic) surfactant. This allows large-pore-size membranes with a high cross-link density and consequently high mechanical strength to be prepared for the separation of large biomolecules.

Electrosynthesis of imprinted polyacrylamide membranes for the stereospecific l-histidine sensor and its characterization by AC impedance spectroscopy and piezoelectric quartz crystal technique

l-Histidine/ d-histidine-imprinted membranes have been synthesized by electropolymerizing acrylamide onto Au electrodes or Au-coated quartz crystal electrodes in the presence of l-histidine/ d-histidine. AC impedance spectroscopy and piezoelectric quartz crystal (PQC) technique were employed to verify the template effect of the membranes. The selectively rebinding of the enantiomers of histidine by their respective imprinted polymer membranes was confirmed. The result may lead to the construction of biomimetic sensors for the stereospecific determination of l-histidine and the chiral separation of histidine.

Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis

Fab fragments isolated from papain digests of monoclonal antibodies have a wide variety of uses in analytical and in both in vivo and in vitro diagnostic applications. A novel, non-affinity method which uses the Gradiflow to purify Fab fragments from the papain digest of a mouse IgG1 anti- c-myc monoclonal antibody is described. The Gradiflow is a preparative electrophoresis instrument that uses polyacrylamide membranes of known pore size to separate proteins in solution in their native state under mild pH conditions by charge or size. The Fab and Fc fragments from the papain digestion were characterized using isoelectric focusing (IEF) and non-reducing SDS–PAGE in conjunction with IEF and Western blot. There were three Fab isoforms with p I between pH 6.5 and 7.4 while the Fc had a range of isoforms between 6.1 and 6.3. Both Fab and Fc fragments had similar M r of 50 kDa. A charge-based purification strategy was developed to obtain a high purity Fab preparation after 10 min, confirmed by Western blot and chemiluminescence analyses. A small quantity of residual undigested IgG1 remained and was removed using a size-based separation. The efficiency of the separation despite the narrow pH range between Fab and Fc suggests that this technique may be an alternative to protein A or G affinity separation of Fc and Fab monoclonal antibody fragments from papain digests of monoclonal antibodies.

Carrier ampholyte‐free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures

The field of proteomics requires methods that offer high sensitivity and wide dynamic range. One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF). We have modified a commercial solution IEF instrument, the Rotofor, to prefractionate protein mixtures by carrier ampholyte-free solution IEF. The focusing chamber of the Rotofor was divided into several compartments by polyacrylamide membranes with imbedded Immobiline mixtures of specific pH values. When an electric field is applied, each protein migrates to the compartment confined by membranes with pH values flanking its isoelectric point. The approach was demonstrated for the focusing of myoglobin into a predicted compartment, as well as the separation of a complex soluble yeast protein mixture into several distinct fractions. The proteins were dissolved in water or 30% isopropanol. The method is applicable to both gel-based and solution-phase protein identification methods, without the need for further sample preparation.

Design of a new, twelve-channel electrophoretic apparatus based on the Gradiflow technology

The Gradiflow technology, originally designed to carry out binary, size-based and charge sign-based electrophoretic protein separations, has been extended to simultaneously obtain multiple protein fractions from a single electrophoretic separation. The separation unit of the new apparatus houses the anode and cathode compartments and up to twelve shallow separation compartments through which the background electrolyte solution that contains the separated protein fractions is recirculated. The separation compartments are formed from grids as thin as 1.2 mm and polyacrylamide membranes as thin as 0.15 mm, all with corresponding multiple inlet and outlet ports. The average pore size of the polyacrylamide membranes can be varied to permit passage of proteins in the 5000–800 000 molecular mass range. The electric field, orthogonal to the flow paths of the recirculated background electrolyte, selectively moves the sample components across the polyacrylamide separation membranes. Selective protein transport can be achieved by exploiting differences in either the relative size of the proteins or the charge sign of the proteins. The advantages of the new apparatus stem from the synergistic combination of the short electrophoretic transfer distances, high electric field strength, large effective surface areas of the separation membranes, and the great flexibility with which apparati containing one to twelve separation compartments can be created.

Preparative electrophoresis: a general method for the purification of polyclonal antibodies

Antibodies were purified from normal rabbit, sheep, goat, rat, human and bovine serum using preparative electrophoresis on a Gradiflow in a single-step process using an asymmetrical cartridge with three different pore size polyacrylamide membranes. Recoveries in each case were over 80% and were higher than those obtained using affinity chromatography on protein A, protein G or protein L. Degree of purity was at least comparable with these methods. These results suggest that preparative electrophoresis can be considered a general method for the purification of research quantities of antibodies from multiple serum sources and may be particularly useful where the reactivity with protein A, G or L is unknown.

Purification of fibrinogen and virus removal using preparative electrophoresis.

The Gradiflow is a novel, scalable preparative electrophoresis technique that uses the dual characteristics of size and charge to isolate target macro- and micromolecules from complex biological solutions. It does this with high resolution and in rapid time. The mild buffers are used to assist in retaining biological activity of the isolated protein. Gradiflow technology employs a sandwich of three polyacrylamide membranes configured to allow passage of macromolecules ranging in size from 10 kDa to 1,500 kDa. Fibrinogen was isolated from cryoprecipitate 1 using a single phase process. This separation was achieved within three hours with yields of 85%. Purified fibrinogen was then characterized using biophysical characterization of fibrin clot structure and compared with clots derived from a commercially available product and human plasma. Significantly, clots developed from Gradiflow fibrinogen had characteristics closer to human plasma. Viral removal characteristics of the Gradiflow were investigated by spiking the source material (cryoprecipitate 1) with canine parvovirus and testing for its presence in the isolated fibrinogen using PCR. Parvo removal was found to be greater than 4 logs and was achieved during the purification process. The Gradiflow offers the advantage of large-scale separation of macromolecules and provides a new approach to fibrinogen separation that is quite distinct from other present-day technologies. The technology is capable of isolating protein with high purity, recovery, and functionality in combination with the removal of viruses during the purification. Furthermore, it is capable of integrating into present production systems, significantly improving yield and functionality of target molecules.

Electrophoretic transfer of proteins across polyacrylamide membranes

The electrophoretic transfer of purified proteins has been examined in a Gradiflow “Babyflow BF100” unit. A number of factors affect protein separation within this preparative electrophoresis system. We established that the rate of protein transfer was proportional to the applied voltage. The transfer is slowest at the isoelectric point (p I) and increased the further away the pH was from the p I of the protein. Protein transfer was found to be independent of the ionic strength of the buffer, for buffers that excluded the addition of strong acids or strong bases or sodium chloride. Transfer decreased as the pore size of the membrane decreased. Finally, transfer was inhibited at high salt concentrations in the protein solution, but remained unaffected when urea and non-ionic detergents were added to the solution. To increase the speed of protein separations, buffers with low conductivity should be used. A pH for the optimal separation should be selected on the basis of the relative p I and size of the target proteins and that of the major contaminants.