Stable pH Gradient Research Articles

Exploiting the pH-Cross Sensitivity of Ion-Selective Optodes to Broaden Their Response Range.

While the pH cross-sensitivity of chromoionophore-based ion-selective optodes (ISOs) has often been regarded as a significant limitation, this paper demonstrates how this apparent drawback can be transformed into a beneficial feature. The response range of chromoionophore-based ISOs shifts proportionally with changes in the sample pH. Thus, integrating them with a stable pH gradient across the optode surface, such as those provided by immobilized pH gradient (IPG) gels, allows for significant enhancement of the effective measuring range of chromoionophore-based ISOs while preserving their maximum sensitivity. We show that the measuring range of sodium-selective chromoionophore-based optodes can be increased up to 2.5 log units when used with commercially available IPG gels. This improvement in measuring range is directly correlated with the pH difference in the pH gradient across the optode, suggesting that even greater enhancements are possible with more substantial pH gradients. Furthermore, this approach is not confined to sodium-selective optodes but can be readily adapted to other ion-selective chromoionophore-based optodes, broadening their potential applications and impact in the field of chemical sensing. This work paves the way for the development of more versatile and highly sensitive optodes across a broad range of analytes, leveraging the pH cross-sensitivity as a tool for enhanced performance.

Streamlined integrated protein isoelectric focusing using microfluidic paper-based device

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes.

Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP's interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine's activities in vivo.

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.

Spatiotemporally Resolved pH Measurement in Aerosol Microdroplets Undergoing Chloride Depletion: An Application of In Situ Raman Microspectrometry.

Acidity is a defining property of atmospheric aerosols that profoundly affects environmental systems, human health, and climate. However, directly measuring the pH of aerosol microdroplets remains a challenge, especially when the microdroplets' composition is nonhomogeneous or dynamically evolving or both. As a result, a pH measurement technique with high spatiotemporal resolution is needed. Here, we report a spatiotemporally resolved pH measurement technique in microdroplets using spontaneous Raman spectroscopy. Our target sample was the microdroplets comprising sodium chloride and oxalic acid─laboratory surrogates of sea spray aerosols and water-soluble organic compounds, respectively. Our measurements show that the chloride depletion from the microdroplets caused a continuous increase in pH by ∼0.5 units in 2 hours. Meanwhile, the surface propensity of chloride anions triggers a stable pH gradient inside a single droplet, with the pH at the droplet surface lower than that at the core by ∼ 0.4 units. The uncertainties arising from the Raman detection limit (±0.08 pH units) and from the nonideal solution conditions (-0.06 pH units) are constrained. Our findings indicate that spontaneous Raman spectroscopy is a simple yet robust technique for precise pH measurement in aerosols with high spatiotemporal resolution.

Performance of five commercial bipolar membranes under forward and reverse bias conditions for acid-base flow battery applications

The list of bipolar membrane (BPM) applications is constantly growing, thanks to the ability of BPMs to enhance water dissociation at low voltage losses and maintain a stable pH gradient across the membrane. Despite several BPMs are available in the market, a comprehensive characterization of membrane properties is still lacking, as BPMs are typically tested only via a steady-state polarization sweep under reverse bias. This work aims to present a systematic characterization of five commercial bipolar membranes using different techniques, investigating BPMs in terms of material and electrochemical properties, as well as overall performance in an acid-base flow battery system (i.e., both under reverse and forward bias). In particular, when testing BPMs in a single-cell acid-base flow battery, we reached round-trip efficiencies up to 65% using current densities of 60 A/m2 during discharge and 240 A/m2 during charge. Moreover, this work shows how further research is needed to lower the proton leakage through the anion exchange layer and electrical resistance, to improve the performance of BPMs for acid-base flow battery and other energy applications.

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 15mmol/L Tris-acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9mL/min.

Proton gradients and pH oscillations emerge from heat flow at the microscale

Proton gradients are essential for biological systems. They not only drive the synthesis of ATP, but initiate molecule degradation and recycling inside lysosomes. However, the high mobility and permeability of protons through membranes make pH gradients very hard to sustain in vitro. Here we report that heat flow across a water-filled chamber forms and sustains stable pH gradients. Charged molecules accumulate by convection and thermophoresis better than uncharged species. In a dissociation reaction, this imbalances the reaction equilibrium and creates a difference in pH. In solutions of amino acids, phosphate, or nucleotides, we achieve pH differences of up to 2 pH units. The same mechanism cycles biomolecules by convection in the created proton gradient. This implements a feedback between biomolecules and a cyclic variation of the pH. The finding provides a mechanism to create a self-sustained proton gradient to drive biochemical reactions.

Chemical bias of electrochemical and photoelectrochemical water splitting using a hydrogel separator

Abstract Resorcinol-formaldehyde hydrogels are shown to be adequate separators in electrochemical and photoelectrochemical water splitting cells. Combined with concentrated buffer electrolytes, they allow the maintenance of relatively stable pH gradients between basic anolytes and acidic catholytes. The water splitting potential at a current density of 3.0 mA/cm 2 applied between two Pt electrodes and a pH bias of 8.1 units retained a value of 2.7 V for several hours. Using iron foam/hematite as photoanode and Pt as cathode, the water splitting photocurrents at an applied potential of 1.23 V reached values of 0.40 and 0.06 mA/cm 2 in the presence and absence, respectively, of this chemical bias.

Nuclear proton dynamics and interactions with calcium signaling

Biochemical signals acting on the nucleus can regulate gene expression. Despite the inherent affinity of nucleic acids and nuclear proteins (e.g. transcription factors) for protons, little is known about the mechanisms that regulate nuclear pH (pHnuc), and how these could be exploited to control gene expression. Here, we show that pHnuc dynamics can be imaged using the DNA-binding dye Hoechst 33342. Nuclear pores allow the passage of medium-sized molecules (calcein), but protons must first bind to mobile buffers in order to gain access to the nucleoplasm. Fixed buffering residing in the nucleus of permeabilized cells was estimated to be very weak on the basis of the large amplitude of pHnuc transients evoked by photolytic H+-uncaging or exposure to weak acids/bases. Consequently, the majority of nuclear pH buffering is sourced from the cytoplasm in the form of mobile buffers. Effective proton diffusion was faster in nucleoplasm than in cytoplasm, in agreement with the higher mobile-to-fixed buffering ratio in the nucleus. Cardiac myocyte pHnuc changed in response to maneuvers that alter nuclear Ca2+ signals. Blocking Ca2+ release from inositol-1,4,5-trisphosphate receptors stably alkalinized the nucleus. This Ca2+-pH interaction may arise from competitive binding to common chemical moieties. Competitive binding to mobile buffers may couple the efflux of Ca2+via nuclear pores with a counterflux of protons. This would generate a stable pH gradient between cytoplasm and nucleus that is sensitive to the state of nuclear Ca2+ signaling. The unusual behavior of protons in the nucleus provides new mechanisms for regulating cardiac nuclear biology.

Switchable pH actuators and 3D integrated salt bridges as new strategies for reconfigurable microfluidic free-flow electrophoretic separation

We present novel strategies for reconfigurable, high-throughput microfluidic free-flow electrophoretic separation using electrically switchable pH actuators and 3D integrated salt bridges to allow rapid formation of stable pH gradients and efficient electrophoresis. The pH actuator is achieved by microfluidic integration of bipolar membranes which change electrolyte pH by injecting excess H(+) or OH(-) ions produced by a field-enhanced water dissociation phenomenon at the membrane junction upon voltage bias. The technique does not require conventional multiple buffer inflows and leaves no gas production as experienced in electrolysis, thus providing stable pH gradients for isoelectric focusing (IEF) separation. With the pH actuator inactivated, the platform can perform zone electrophoretic (ZE) separation in a medium of constant pH. We also describe the use of 3D integrated ion conductive polymers that serve as salt bridges for improving the voltage efficiency of electrophoresis and to allow high throughput. The proof of concept was successfully demonstrated for free-flow IEF and ZE separation of protein mixtures showing the potential and the simplicity of the platform for high-throughput and high-precision sample separation.

Changes in H+-ATPase activity of plasma membrane-enriched vesicles isolated from physic nut roots and leaves of energy and medicinal plant, Jatropha curcas L., as an adaptation mechanism to salinity

The plasma membrane H+-ATPase (PM H+-ATPase) plays a key role in the activation of ion and nutrient transport, and is proposed to be involved in salt tolerance. This study investigates the acclimation of PM H+-ATPase in physic nut (Jatropha curcas L.) roots and leaves treated with 0, 50 and 200 mM NaCl for 5 days. Upon comparison with control roots, the H+-ATPase hydrolytic activity, Vmax, Km, H+-pumping activity and pH gradient potential across the plasma membrane were significantly higher in roots treated with NaCl, especially under mild salt stress. The translational activation of PM H+-ATPase of physic nut roots helped to maintain K+ and Ca2+ uptake as well as nitrogen and phosphorus uptake in roots. Compared with the control leaves, the maintenance of H+ transport under 50 mM NaCl was attributed to a modification of the lipid membrane, and the increase in H+transport under 200 mM NaCl could be because of an acid-mediated activation. Differential expression of the H+-ATPase isoforms could also occur in leaves under salt stress. Thus, the stable pH gradient created by the H+-ATPase in leaves treated with 50 mM NaCl assisted normal leaf growth. These modulations of PM H+-ATPase in the roots and leaves of physic nuts could represent a set of adaptive mechanisms to salinity. Key words: Immunoprecipitation, Jatropha curcas L., plasma membrane H+-ATPase, salt stress.

Confocal Raman Microscopy of pH-Gradient-Based 10 000-Fold Preconcentration of Compounds within Individual, Optically Trapped Phospholipid Vesicles

A stable pH gradient established across the membrane of phospholipid vesicle can induce the accumulation of ionizable compounds from bulk solution into the vesicle interior. This pH-gradient vesicle loading process has previously been utilized to encapsulate drugs in pharmaceutical liposomal formulations. In the present work, this process is exploited to preconcentrate dilute analytes from free solution into phospholipid vesicles, which are then detected by optically trapping individual vesicles and measuring their contents using confocal Raman microscopy. The theory of accumulation, based on the acid-base ionization equilibria of the analyte, is developed to account for depletion of the source phase and the finite buffering capacity of the vesicle interior. The model predicts that, under appropriate conditions, enrichment factors of more than 4 orders of magnitude can be realized. To test the concept, experiments were performed measuring the accumulation of benzyldimethylamine into 600-nm phospholipid vesicles. Manipulation of vesicles by optical trapping allows accumulation within an individual vesicle to be characterized while varying the external solution conditions. A more than 10 000-fold enrichment of the analyte concentration inside the vesicle relative to the source phase is reported. The results suggest that pH-gradient loading could be exploited as a powerful preconcentration scheme for trace analysis using either Raman microscopy or other microspectroscopy techniques.

Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii

The microalgae Chlamydomonas reinhardtii and Chlorella sp. CCAP 211/84 were grown autotrophically and mixotrophically and their thermoluminescence emissions were recorded above 0 °C after excitation by 1, 2 or 3 xenon flashes or by continuous far-red light. An oscillation of the B band intensity according to the number of flashes was always observed, with a maximum after 2 flashes, accompanied by a downshift of the B band temperature maximum in mixotrophic compared to autotrophic grown cells, indicative of a dark stable pH gradient. Moreover, new flash-induced bands emerged in mixotrophic Chlamydomonas grown cells, at temperatures higher than that of the B band. In contrast to the afterglow band observed in higher plants, in Chlamydomonas these bands were not inducible by far-red light, were fully suppressed by 2 μM antimycin A, and peaked at different temperatures depending on the flash number and growth stage, with higher temperature maxima in cells at a stationary compared to an exponential growth stage. These differences are discussed according to the particular properties of cyclic electron transfer pathways in C. reinhardtii.

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.

Heat-activated sustaining nitric oxide release from zwitterionic diazeniumdiolate loaded in thermo-sensitive liposomes

Zwitterionic diazeniumdiolate, a nitric oxide precursor, was dissolved in basic buffer solution (pH = 9.0) and encapsulated in thermo-sensitive liposomes composed of phospholipids of different sensitive temperatures. The basic intra-liposomal environment dramatically delayed spontaneous NO release. When the liposomes were placed in physiological buffer solution and temperatures were increased to the sensitive temperatures of the phospholipids’ membranes, extra-liposomal protons started to influx to collapse the pH gradient and subsequently induce a significant NO release. Moreover, the presence of a stronger pH gradient when the liposomes were applied to a more acidic environment caused a higher proton influx driving force to trigger the influx of protons and, subsequently, NO release. In this work, we demonstrate that thermo-sensitive liposomes can be used to create a stable pH gradient in a nanoenvironment entrapping zwitterionic diazeniumdiolate for sustained and heat/acid-activated NO release.

In situ generation of pH gradients in microfluidic devices for biofabrication of freestanding, semi-permeable chitosan membranes

We report the in situ generation of pH gradients in microfluidic devices for biofabrication of freestanding, semi-permeable chitosan membranes. The pH-stimuli-responsive polysaccharide chitosan was enlisted to form a freestanding hydrophilic membrane structure in microfluidic networks where pH gradients are generated at the converging interface between a slightly acidic chitosan solution and a slightly basic buffer solution. A simple and effective pumping strategy was devised to realize a stable flow interface thereby generating a stable, well-controlled and localized pH gradient. Chitosan molecules were deprotonated at the flow interface, causing gelation and solidification of a freestanding chitosan membrane from a nucleation point at the junction of two converging flow streams to an anchoring point where the two flow streams diverge to two output channels. The fabricated chitosan membranes were about 30-60 microm thick and uniform throughout the flow interface inside the microchannels. A T-shaped membrane formed by sequentially fabricating orthogonal membranes demonstrates flexibility of the assembly process. The membranes are permeable to aqueous solutions and are removed by mildly acidic solutions. Permeability tests suggested that the membrane pore size was a few nanometres, i.e., the size range of antibodies. Building on the widely reported use of chitosan as a soft interconnect for biological components and microfabricated devices and the broad applications of membrane functionalities in microsystems, we believe that the facile, rapid biofabrication of freestanding chitosan membranes can be applied to many biochemical, bioanalytical, biosensing applications and cellular studies.

Microchip free flow isoelectric focusing for protein prefractionation using monolith with immobilized pH gradient

Microchip free flow IEF (muFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 mum depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional muFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled beta-lactoglobulin and carbonic anhydrase, with 0.9 unit difference on pI, was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of muFFIEF with M-IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.

Free-flow isoelectric focusing microfluidic device with glass coating by sol–gel methods

A device for microfluidic free-flow isoelectric focusing (μ-FFIEF) was fabricated in a glass-coated PDMS structure. The microfluidic channel was made by a PDMS replica molding and standard soft photolithography technique. For the prefractionation and preconcentration of a protein mixture, we patterned palladium electrodes on the glass substrate and coated with TEOS–sol to enhance the bonding with PDMS. The PDMS microchannel was coated with glass by flushing TEOS–sol and annealing at 100 °C. The optimum focusing condition was obtained by pH indicator with ampholyte solution made with MES. Model proteins, RFP and EGFP, were successfully concentrated around 214 μm and 357 μm from the anode, respectively. The optimum focusing condition in the μ-FFIEF device was an electric potential of 1.5 V/cm and pH 6.0. The focusing time in the channel without glass coating was approximately 30 s, but the breakdown of the electrode by electrolysis started immediately when the electric field was applied. However, the glass-coated device was kept intact under continuous application of voltage giving a stable pH gradient, but with twice the focusing time.

High-performance protein separation by ion exchange membrane partitioned free-flow isoelectric focusing system

The objective of this study is to investigate a combination of ion exchange membranes and free-flow isoelectric focusing (FFIEF) technology for high-selectivity and high-flux protein separation, in which ion exchange membranes are used as the separation media. An FFIEF device has been designed and extensive experiments have been conducted to prove its effectiveness in enhancing the protein separation performance. Three types of membranes were employed in this work and they were commercial microfiltration (MF) ion exchange membranes, commercial neutral UF cellulose membranes, and home-made ultrafiltration sulfonated polysulfone (UF SPSf) ion exchange membranes. The protein separation results show that the home-made UF SPSf membranes have the superior selectivity and flux to other membranes. This is due to the fact that a stable pH gradient across the membranes as well as the interaction between the protein molecules and membrane surface plays an important role in the high-performance protein separation. By applying a semi-batch separation process and optimizing various experimental conditions, a high-purity ( > 90 % ) and concentrated target protein is obtained at the permeation side of the home-made UF SPSf membranes with a high flux.