Generation Of pH Gradient Research Articles

Electrochemical Conversion of Bicarbonate Using Proton Exchange Membrane-Based Electrolyzer

Direct electrochemical conversion of captured carbon dioxide (CO2) is gaining interest as an efficient method for capturing and utilizing CO2, eliminating the need for energy-intensive CO2 separation process.[1] The integrated electrolyzer with bicarbonate feed operates on the principle of spontaneous reduction of CO2 released from bicarbonate within the electrolyzer. The key to the on-site decomposition of bicarbonate into CO2 is to acidify the bicarbonate within the electrolyzer through the deliberately generated pH gradient. Bipolar membrane has been extensively used to generate pH gradient and acidify the bicarbonate feed.[2] However, the high ionic resistivity of bipolar membrane and slow kinetic of water dissociation reaction can result in a high operation cell voltage.In this study, we demonstrate proton exchange membrane based-bicarbonate electrolyzer which produces carbon monoxide from bicarbonate feedstock with high CO production selectivity and low cell voltage that are comparable to the conventional gas fed CO2 electrolyzer. To achieve highly selective CO production selectivity, a nickel single atom catalyst (Ni SAC) where the atomically dispersed nickel active sites decorated on the nitrogen doped carbon was prepared and utilized. Ni-SAC showed high CO production selectivity even in bicarbonate feed electrolyzer. To further improve the performance of this integrated electrolyzer, significant attention was given to the optimization of electrode structures. Due to the complexity of the reaction environment within the integrated electrolzyer, the performance of the electrolyzer is heavily dependent on the porosity of electrodes and distribution of catalyst on the electrodes. By optimizing the fabrication of electrodes with Ni-SACs, the optimal catalyst coated electrode showed high CO production selectivity over 90% with current density of 100 mA/cm2 at low operating cell potential of 3 V.

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.

Electrolytic Regeneration of Spent Caustic Soda from CO2 Capture Systems

The traditional electrochemical caustic soda recovery system uses the generated pH gradient across the ion exchange membrane for the regeneration of spent alkaline absorbent from CO2 capture. This electrochemical CO2 capture system releases the by-products H2 and O2 at the cathode and anode, respectively. Although effective for capturing CO2, the slow kinetics of the oxygen evolution reaction (OER) limit the energy efficiency of this technique. Hence, this study proposed and validated a hybrid electrochemical cell based on the H2-cycling from the cathode to the anode to eliminate the reliance on anodic oxygen generation. The results show that our lab-scale prototype enabled effective spent caustic soda recovery with an electron utilisation efficiency of 90%, and a relative carbonate/bicarbonate diffusional flux of approximately 40%. The system also enabled the regeneration of spent alkaline absorbent with a minimum electrochemical energy input of 0.19 kWh/kg CO2 at a CO2 recovery rate of 0.7 mol/m2/h, accounting for 30% lower energy demand than a control system without H2-recycling, making this technique a promising alternative to the conventional thermal regeneration technology.

Cyclic AMP signalling and glucose metabolism mediate pH taxis by African trypanosomes

The collective movement of African trypanosomes on semi-solid surfaces, known as social motility, is presumed to be due to migration factors and repellents released by the parasites. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising properties on agarose plates. While early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. Furthermore, trypanosomes respond to exogenously formed pH gradients, with both early and late procyclic forms being attracted to alkali. pH taxis is mediated by multiple cyclic AMP effectors: deletion of one copy of adenylate cyclase ACP5, or both copies of the cyclic AMP response protein CARP3, abrogates the response to acid, while deletion of phosphodiesterase PDEB1 completely abolishes pH taxis. The ability to sense pH is biologically relevant as trypanosomes experience large changes as they migrate through their tsetse host. Supporting this, a CARP3 null mutant is severely compromised in its ability to establish infections in flies. Based on these findings, we propose that the expanded family of adenylate cyclases in trypanosomes might govern other chemotactic responses in their two hosts.

Electrical Writing to Three-Dimensional Pattern Dynamic Polysaccharide Hydrogel for Programmable Shape Deformation.

The ability to pattern and actuate hydrogels is essential for biomimetics, soft robotics, and biosensors. Here an electrical writing technique with the capability to create both surface and across thickness patterns in dynamic chitosan-H+ /agarose hydrogel by electronically generated pH gradient is introduced. The diffusible pH cues deprotonate and re-assemble chitosan chains by hydrogen bonds, changing the electrical writing domains from original loose structure to a dense layer and resulting in different mechanical stress and swell ability that causes the hydrogel to deform. The deformable trend can be modulated by writing depth and selective writing area on the surface, and significantly enhanced by temperature increment. Finally, a dual electrical writing process to create three-dimensional patterns and demonstrate programmable shape transition by differing patterns is performed.

Controllable pH Manipulations in Micro/Nanofluidic Device Using Nanoscale Electrokinetics.

Recently introduced nanoscale electrokinetic phenomenon called ion concentration polarization (ICP) has been suffered from serious pH changes to the sample fluid. A number of studies have focused on the origin of pH changes and strategies for regulating it. Instead of avoiding pH changes, in this work, we tried to demonstrate new ways to utilize this inevitable pH change. First, one can obtain a well-defined pH gradient in proton-received microchannel by applying a fixed electric current through a proton exchange membrane. Furthermore, one can tune the pH gradient on demand by adjusting the proton mass transportation (i.e., adjusting electric current). Secondly, we demonstrated that the occurrence of ICP can be examined by sensing a surrounding pH of electrolyte solution. When pH > threshold pH, patterned pH-responsive hydrogel inside a straight microchannel acted as a nanojunction to block the microchannel, while it did as a microjunction when pH < threshold pH. In case of forming a nanojunction, electrical current significantly dropped compared to the case of a microjunction. The strategies that presented in this work would be a basis for useful engineering applications such as a localized pH stimulation to biomolecules using tunable pH gradient generation and portable pH sensor with pH-sensitive hydrogel.

Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors.

Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (IF) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (IR) remained constant at any pH. The ratio of IF/IR produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.

Tumor-on-a-chip: a microfluidic model to study cell response to environmental gradients.

Limited blood supply and rapid tumor metabolism within solid tumors leads to nutrient starvation, waste product accumulation and the generation of pH gradients across the tumor mass. These environmental conditions modify multiple cellular functions, including metabolism, proliferation, and drug response. However, capturing the spatial metabolic and phenotypic heterogeneity of the tumor with classic in vitro models remains challenging. Thus, in this work a microfluidic tumor slice model was developed to study cell behavior under metabolic starvation gradients. The presented microdevice comprises a central chamber where tumor cells were cultured in a 3D collagen hydrogel. A lumen on the flank of the chamber was used to perfuse media, mimicking the vasculature. Under these circumstances, tumor cell metabolism led to the generation of viability, proliferation and pH gradients. The model decoupled the influence of oxygen from other nutrients, revealing that cell necrosis at the core of the model could be explained by nutrient starvation. The microdevice can be disassembled to retrieve the cells from the desired locations to study molecular adaptions due to nutrient starvation. When exposed to these pH gradients and low nutrient conditions, cancer cells showed multiple changes in their gene expression profile depending on their distance from the lumen. Those cells located further from the lumen upregulated several genes related to stress and survival response, whereas genes related to proliferation and DNA repair were downregulated. This model may help to identify new therapeutic opportunities to target the metabolic heterogeneity observed in solid tumors.

(Invited) Particulate Photocatalyst Systems for Sunlight-Driven Water Splitting

Sunlight-driven water splitting has been studied actively for production of renewable solar hydrogen as a storable and transportable energy carrier [1-3]. Both the efficiency and the scalability of water-splitting systems are important factors for practical utilization of renewable solar hydrogen because of the low areal density of solar energy. Particulate photocatalyst systems do not involve any secure electric circuit and thus can be spread over a wide area by inexpensive processes potentially. In this regard, activation of particulate photocatalysts and development of their reaction systems are important subjects. A semiconductor photocatalyst can split water into hydrogen and oxygen thermodynamically when the band gap straddles the potentials of the hydrogen evolution reaction (0 V vs. RHE) and the oxygen evolution reaction (+1.23 V vs. RHE). In addition, it is generally necessary to modify photocatalysts with appropriate cocatalysts in order to facilitate charge separation and surface redox reactions. The authors’ group has studied various semiconducting materials including oxides, (oxy)nitrides, and (oxy)chalcogenides for photocatalytic water splitting [1]. Recently, we have found that doping Al into SrTiO3 boosts the water splitting activity by two orders of magnitude [4]. The resultant Al-doped SrTiO3 achieved an apparent quantum yield (AQY) of 30% at 360 nm. Through the optimization of the preparation and modification methods of Al-doped SrTiO3, the AQY of photocatalytic water splitting has been upgraded to 56% and higher at 365 nm. Processing of such particulate photocatalysts into potentially extensible forms is to be presented [5]. Two different photocatalysts can also be combined so that hydrogen and oxygen are generated on the different photocatalysts [1,3]. Recently, the authors’ group has developed particulate photocatalyst sheets consisting of the hydrogen evolution photocatalyst (HEP) and the oxygen evolution photocatalyst (OEP) embedded into conductive layers by particle transfer [6-11]. A photocatalyst sheet consisting of La- and Rh-codoped SrTiO3 as the HEP and Mo-doped BiVO4 as the OEP embedded into a carbon conductor exhibits a solar-to-hydrogen energy conversion efficiency of 1.0% at ambient pressure [10]. The photocatalyst sheet shows significantly higher water splitting activity than the corresponding powder suspension systems, because the conductor layer transfers photogenerated electrons between photocatalyst particles effectively. In addition, evolution of hydrogen and oxygen in close proximity allows to prevent generation of pH gradient during the water splitting reaction. Therefore, the photocatalyst sheet is scalable directly without sacrificing the high activity. However, the absorption edge wavelengths of La- and Rh-codoped SrTiO3 and Mo-doped BiVO4 are 520 nm at most. It is necessary to utilize photocatalysts with longer absorption edge wavelengths to pursuit higher STH values. We have found that some (oxy)chalcogenides and (oxy)nitrides with narrower band gap energies are applicable as the HEP and the OEP of particulate photocatalyst sheets. In this talk, recent progress and future challenges in photocatalytic water splitting and system development will be presented. Hisatomi et al., Chem. Soc. Rev. 2014, 43, 7520.Hisatomi et al., Catal. Lett. 2015, 145, 95.Hisatomi et al., Faraday Discuss. 2017, 198, 11.Ham et al., J. Mater. Chem. A 2016, 4, 3027.Xiong et al., Catal. Sci. Technol. 2014, 4, 325.Minegishi et al., Chem. Sci. 2013, 4, 1120.Wang et al., J. Catal. 2015, 328, 308Wang et al., Nat. Mater. 2016, 15, 611Wang et al., Faraday Discuss. 2017, 197, 491Wang et al., J. Am. Chem. Soc. 2017, 139, 1675.Hisatomi et al., Curr. Opin. Electrochem. 2017, 2, 148.

A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins.

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro‐column, pH gradient generation, and fraction collection in a single device. Using a sieve micro‐valve, anion exchange particles were packed into a microchannel in order to realize a solid‐phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro‐column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin–fluorescein isothiocyanate conjugate (FITC‐BSA) and R‐Phycoerythrin (R‐PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.

(Keynote) Development of Particulate Photocatalyst Systems for Efficient and Scalable Water Splitting

Water splitting driven by solar energy has attracted much attention as a means of renewable solar hydrogen production. Photocatalytic water splitting is a representative approach utilizing particulate semiconductors.1,2 When a particulate semiconductor photocatalyst absorbs a photon, an electron is excited from the valence band to the conduction band, and a positive hole is left in the valence band. These excited electrons and holes can drive reduction and oxidation reactions on each photocatalyst particle, respectively. A photocatalyst can split water into hydrogen and oxygen thermodynamically when the band gap straddles the potentials of the hydrogen evolution reaction (0 V vs. RHE) and the oxygen evolution reaction (+1.23 V vs. RHE); however, to drive the reaction efficiently, it is generally necessary to load nanoparticulate metals and metal oxides, denoted as cocatalysts, on the photocatalyst as active sites for the hydrogen evolution reaction and the oxygen evolution reaction. Note that it is also possible to split water using two kinds of photocatalysts for hydrogen evolution and oxygen evolution, respectively. Such a reaction scheme based on two-step excitation is called Z-scheme. A solar-to-hydrogen energy conversion efficiency (STH) of 5% or higher is considered to be necessary for practical operation of photocatalytic solar hydrogen production. To attain such a high STH at reasonable quantum efficiencies, it is necessary to activate and stabilize narrow band gap photocatalysts.2 Scalability of the system is also a critical issue.3 Water splitting systems based on particulate photocatalysts are promising in terms of scalability because they do not need any secure electric circuits and thus can be spread over a wide area easily. Conventionally, photocatalytic water splitting has been studied using suspension of particulate photocatalysts. However, this approach is not feasible in practice because it is challenging to design a cheap photocatalytic reactor that can maintain a large amount of water over a wide area. It is probably a more feasible to use photocatalysts processed into panels and construct modules similar to photovoltaic applications.4 This approach, fixing particulate photocatalysts onto substrate, is also expected to be advantageous over the conventional powder suspension approach in operation and maintenance, because power input to stir a large amount of photocatalyst suspension is unneeded and used photocatalysts can be readily replaced with fresh ones. Recently, the authors’ group developed photocatalyst sheets based on particulate hydrogen evolution photocatalyst (HEP) and oxygen evolution photocatalyst (OEP) embedded into conductive layers by particle transfer for efficient and scalable water splitting (Figure).5–8 The STH of water splitting using photocatalyst sheets consisting of La- and Rh-codoped SrTiO3 (SrTiO3:La,Rh) as a HEP and BiVO4 as an OEP was 0.2%, superior to those for the corresponding powder suspension systems.6 The photocatalyst sheet maintained the high water splitting activity over a wide range of pH values and even in pure water. Moreover, the STH was improved to 1.1% through improvements in the preparation process of the photocatalyst sheet and the reaction conditions.7 The high activity of the photocatalyst sheet is due to the efficient electron transfer between HEP and OEP particles via the underlying conductive layer. In addition, evolution of hydrogen and oxygen in close proximity allows to prevent generation of pH gradient during the water splitting reaction.8 Therefore, the photocatalyst sheet is scalable directly without sacrificing the high activity. However, the absorption edge wavelengths of SrTiO3:La,Rh and BiVO4 are 540 nm at most. It is still important to develop photocatalysts with longer absorption edge wavelengths. In this talk, recent progress and future challenges in photocatalytic water splitting and system development will be presented. References Hisatomi et al., Chem. Soc. Rev. 2014, 43, 7520. Hisatomi et al., Catal. Lett. 2015, 145, 95. Maeda et al., J. Phys. Chem. Lett. 2010, 1, 2655. Xiong et al., Catal. Sci. Technol. 2014, 4, 325. Minegishi et al., Chem. Sci. 2013, 4, 1120. Wang et al., J. Catal. 2015, 328, 308. Wang et al., Nat. Mater. 2016, 15, 611. Wang et al., Faraday Discuss., in press. Figure 1

Mixed-Beds of Strong and Weak Anion Exchange Resins for Protein Separations with Step-Induced pH Gradients

A new way of performing pH gradient-based protein separations using a mixed-bed column comprising a small-pore weak base resin and a large-pore strong base resin and a step change in pH at the column entrance is introduced. The approach effectively decouples intracolumn generation of pH gradients, which ensues from ionic interactions with the weak base resin, from protein separation, which ensues from interactions with the strong base resin ligands, resulting in a process that is more easily predictable, less prone to the effects of protein loading on the pH gradient, less susceptible to hydrophobic interactions between protein and resin surface, and more flexible. The approach is demonstrated successfully using a tertiary amine resin as the weak base component together with a range of commercial strong anion exchangers with quaternary amine functionality. A predictive model, based on the potentiometric titration of the weak base resin, is in excellent agreement with experimentally determined pH profiles obtained using mixed-bed columns and amine buffers with pH steps from 9 to 7. The separation of protein charge variants with the mixed-bed concept is demonstrated using these columns for a deamidated monoclonal antibody mixture, which yielded highly homogeneous fractions.

Modeling of salt and pH gradient elution in ion-exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

New solution IEF device for micropreparative separation of peptides and proteins

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.

Improving selectivity in multimodal chromatography using controlled pH gradient elution

Externally generated pH gradients are employed on a multimodal cation exchange chromatographic resin to improve the selectivity for a mixture of model proteins. By combining controlled pH gradients with the unique selectivities arising from the multiple interaction types exhibited by the multimodal resin, the separation of the protein mixture is significantly improved as compared to linear salt gradient operation. Several gradient conditions are explored and a shallow gradient from pH 3.8 to 5.5 is shown to be able to resolve the proteins. This work provides proof of concept for the use of pH gradients in multimodal chromatography and sets the stage for future applications.

Fabrication of an optically driven pH gradient generator based on self-assembled proton pumps

Optically manipulating the local pH of a target solution in a microchannel, or reservoir, provides a mechanism for activating and controlling a variety of biological and chemical processes on Lab-on-a-Chip (LOC) devices and micro-total analysis systems (μ-TAS). A microscale pH gradient generator that exploits the light-activated molecular proton pumps found in the purple membranes (PM) of bacteriorhodopsin is described in this paper. The photo-electro-chemical transducer is an ultrathin layer (~13 nm) of oriented PM patches self-assembled on an Au-coated porous substrate. A biotin labeling and streptavidin molecular recognition technique is used to ensure that the extracellular side of all PM patches is attached to the porous substrate enabling unidirectional and efficient transport of ions across the transducer surface. The photo-induced proton pumps generate a flow of ions that produce a measurable change in pH between the separated solutions. The self-assembly procedure is experimentally quantified based on the capacitance characteristics of the bR membranes. The investigation confirms that the transducer is covered with the bR proton pumps at a mass density of 2.33 ng/cm2. Experimental tests also show that the proposed transducer can repeatedly generate pH gradients as high as 0.42 and absolute voltage differences as high as 25 mV when illuminated by an 18 mW, 568 nm light source. Furthermore, the ΔpH is observed to be nonlinear with respect to light intensity and exposure time. The ΔpH of the target solution is sufficient to cause a phenolphthalein indicator dye to change color or an ionic hydrogel micro-valve to expand.

Mechanism of anodic electrodeposition of calcium alginate

Stimuli-responsive polysaccharides that can undergo a sol–gel transition in response to localized electrical signals provide a unique opportunity to electroaddress biological components at device interfaces. Most polysaccharide electroaddressing mechanisms use electrochemical reactions to generate pH gradients that can locally neutralize the polysaccharide and induce its reversible sol–gel transition to form a hydrogel film adjacent to the electrode surface. The calcium-responsive polysaccharide alginate is an exception; it may electrodeposit without requiring extreme pH gradients and thus may provide a means to electroaddress pH-sensitive biological components. Here, we use a novel device to characterize the mechanism for the anodic electrodeposition of a calcium alginate hydrogel. This device consists of a transparent fluidic channel with built-in sidewall electrodes that allows Ca–alginate electrodeposition to be directly measured by non-destructive optical and spectroscopic methods. We hypothesize a 3-step mechanism for calcium–alginate electrodeposition: (i) water is electrolyzed to locally generate protons (or hydronium ions); (ii) these protons are consumed by reacting with suspended CaCO3 particles and this “buffering” reaction generates a gradient in soluble Ca2+; and (iii) the locally generated Ca2+ ions interact with alginate to induce its sol–gel transition. We verified this electrodeposition mechanism using pH-responsive dyes to observe the local pH gradients during gel formation, Ca2+ indicator dyes to observe the Ca2+ gradient, and in situ Raman spectroscopy to demonstrate a strong interaction between soluble Ca2+ and alginate. Importantly, these results demonstrate electrodeposition without the need for a substantial pH excursion from neutrality. Thus, calcium alginate appears especially well-suited for electroaddressing labile biological components for applications in biosensors, biofabrication and BioMEMS.

Microscale pH gradient generation by electrolysis on a light-addressable planar electrode

This paper describes a light-addressable electrolytic system to produce microscale pH gradients. Light illumination to a photoconductive planar electrode generates a conducting point serving as a photo-anode or cathode, where applied positive or negative voltage electrolytically produces protons or hydroxide ions leading to an increased or decreased pH gradient. A digital micromirror device (DMD) was installed in this system to provide a patterned light illumination. This flexible spatial addressability is a great advantage over previous on-chip pH gradient generation systems with metal thin film microelectrodes. The fluorescence pH imaging showed a proof of principle that electrolysis at the addressed photo-anodes and cathodes could generate localized pH gradients. The spatio-temporal patternability of photo-electrodes allowed characterization of various gradient profiles without any re-fabrication of electrode patterns, a prerequisite in conventional electrolytic generation with metal thin film microelectrodes. A spatio-temporal switching between the photo-anode and cathode allowing use of a combination of bimodal electrolysis enabled the fine-tuning of gradient profiles and enriched the variety of available gradient patterns.

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.