Bipolar electrode focusing: faradaic ion concentration polarization

Bipolar electrode (BPE) focusing locally enriches charged analytes in a microchannel along an electric field gradient that opposes a counter-flow. This electric field gradient forms at the boundary of an ion depletion zone generated by the BPE. Here, we demonstrate concentration enrichment of a fluorescent tracer by up to 500,000-fold. The use of a dual-channel microfluidic configuration, composed of two microchannels electrochemically connected by a BPE, enhances the rate of enrichment (up to 71-fold/s). Faradaic reactions at the ends of the BPE generate ion depletion and enrichment zones in the two, separated channels. This type of device is equivalent to previously reported micro/nanochannel junction arrangements used for ion concentration polarization, but it is experimentally more flexible and much simpler to construct.     

Coupled electrochemical reactions at bipolar microelectrodes and nanoelectrodes

Here we report the voltammetric study of coupled electrochemical reactions on microelectrodes and nanoelectrodes in a closed bipolar cell. We use steady-state cyclic voltammetry to discuss the overall voltammetric response of closed bipolar electrodes (BPEs) and understand its dependence on the concentration of redox species and electrode size. Much of the previous work in bipolar electroanalytical chemistry has focused on the use of an "open" cell with the BPE located in an open microchannel. A closed BPE, on the other hand, has two poles placed in separate compartments and has remained relatively unexplored in this field. In this work, we demonstrated that carbon-fiber microelectrodes when backfilled with an electrolyte to establish conductivity are closed BPEs. The coupling between the oxidation reaction, e.g., dopamine oxidation, on the carbon disk/cylinder and the reduction of oxygen on the interior fiber is likely to be responsible for the conductivity. We also demonstrated the ability to quantitatively measure voltammetric properties of both the cathodic and anodic poles in a closed bipolar cell from a single cyclic voltammetry (CV) scan. It was found that "secondary" reactions such as oxygen reduction play an important role in this process. We also described the fabrication and use of Pt bipolar nanoelectrodes which may serve as a useful platform for future advances in nanoscale bipolar electrochemistry.     

Enrichment of Cations via Bipolar Electrode Focusing

We have previously demonstrated up to 5 × 10(5)-fold enrichment of anionic analytes in a microchannel using a technique called bipolar electrode focusing (BEF). Here, we demonstrate that BEF can also be used to enrich a cationic fluorescent tracer. The important point is that chemical modification of the microchannel walls enables reversal of the electroosmotic flow (EOF), enabling cations, instead of anions, to be enriched via an electric field gradient focusing mechanism. Reversal of the EOF has significant consequences on the formation and shape of the region of the buffer solution depleted of charge carriers (depletion zone). Electric field measurements and numerical simulations are used to elucidate the factors influencing the depletion zone. This information is used to understand and control the location and shape of the depletion zone, which in turn influences the stability and concentration of the enriched band.     

Another approach toward over 100,000-fold sensitivity increase in capillary electrophoresis: electrokinetic supercharging with optimized sample injection

Electrokinetic supercharging (EKS) is a powerful and practical method for multifold in-line concentration of various analytes prior to capillary electrophoresis (CE) analysis. However, a problem of insufficient sensitivity has always existed when trace analyte quantification by EKS-CE is a target, especially when coupled with conventional detectors. Normally this requires a greatly increased amount of analyte injected without separation degradation. In this contribution, we have shown that it is possible to substantially improve analyte loading and hence CE method detectability by modifying sample introduction configuration. The volume of sample vial was increased (from typical 500 μL to 17 mL), the common wire electrode was replaced by a ring electrode, and the sample solution was stirred. With these alterations, more analyte ions are accumulated within the effective electric field during electrokinetic injection and then maintained as focused zones due to transient isotachophoresis. The versatility of the customized EKS-CE approach for sample concentration was demonstrated for a mixture of seven rare-earth metal ions with an enrichment factor of 500?000 giving detection limits at or below 1 ng/L. These detection limits are over 100?000 times better than can be achieved by normal hydrodynamic injection, 1000 times better than the sensitivity thresholds of inductively coupled plasma atomic emission spectrometry (ICP-AES), and even close to those of inductively coupled plasma mass spectrometry (ICPMS).     

Temperature gradient focusing with field-amplified continuous sample injection for dual-stage analyte enrichment and separation

We describe the serial combination of temperature gradient focusing (TGF) and field-amplified continuous sample injection (FACSI) for improved analyte enrichment and electrophoretic separation. TGF is a counterflow equilibrium gradient method for the simultaneous concentration and separation of analytes. When TGF is implemented with a low conductivity sample buffer and a (relatively) high conductivity separation buffer, a form of sample enrichment similar to field-amplified sample stacking (FASS) or field-amplified sample injection (FASI) is achieved in addition to the normal TGF sample enrichment. FACSI-TGF differs from FASI in two important respects: continuous sample injection, versus a discrete injection, is utilized; because of the counterflow employed for TGF, the stacking interface exists in a pseudo-stationary region outside of the separation column. Notably, analyte concentration enrichment factors greater than the ratio of separation and sample conductivities (gamma) were achieved in this method. For gamma=6.1, the concentration factor for one model analyte (Oregon Green 488) was found to be 36-fold higher with FACSI-TGF as compared to TGF without FACSI. A separation of five fluorescently labeled amino acids is also demonstrated with the technique, yielding an average enrichment of greater than 1000-fold.     

Cyclic chronopotentiometry as a detection tool for flowing solution systems

Cyclic chronopotentiometry provides a very simple detection method, which may be particularly useful in capillary electrophoresis (CE) and microseparation systems. It has been shown that for disk microelectrodes it is possible to define safe reduction and oxidation currents that would never lead to the formation of H2 or O2 gas bubbles, even if they are applied for an indefinitely long time period. During end-column CE detection, currents passing through the working microelectrode can be completely controlled by the external electronic circuit and they are not affected by the separation current. Consequently, problems created by the offset potential in CE can be completely eliminated. The detection can be accomplished through a variety of different mechanisms; however, generation of the electrode response as a result of analyte adsorption seems to be most common. The method is applicable to many analytes, which do not have to be electroactive. The analytical signal is obtained by monitoring the change in the average electrode potential (calculated for either a cathodic or an anodic half-cycle) caused by an analyte interacting with the electrode. The analytical signal is proportional to the analyte concentration, within a concentration range extending over approximately 2 orders of magnitude.     

Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis

A method for determining the accurate effective mobility value of an analyte in the presence of a charged interacting agent, such as a charged cyclodextrin, a micellar agent, a protein, or a DNA fragment that binds the traditional electroosmotic flow markers, is presented. Part of the capillary is filled with the charged interacting agent-containing background electrolyte; the other part is filled with the charged interacting agent-free background electrolyte. The analyte band is placed in the charged interacting agent-containing background electrolyte zone, while a neutral marker (electroosmotic flow marker) is placed in the adjacent charged interacting agent-free background electrolyte zone. The initial, preelectrophoresis distance between the analyte band and the neutral marker band is determined by pressure mobilizing the bands past the detector and recording the detector trace. Subsequently, by applying reverse pressure, the bands are moved back into the first portion of the capillary and a brief electrophoretic separation is carried out. Then, the bands are pressure mobilized again past the detector to obtain their final, postelectrophoresis distance. If (i) the neutral marker does not come into contact with the charged interacting agent and (ii) the analyte does not migrate out of the homogeneous portion of the charged interacting agent zone, the accurate effective electrophoretic migration distance of the analyte, corrected for bulk flow transport, can be determined. The actual electric field strengths in the different zones of the heterogeneously filled capillary can be calculated from the integral of the electrophoretic current and the conductivity of the charged interacting agent-containing background electrolyte measured in a separate experiment. Once the effective mobility of an analyte in the charged resolving agent-containing background electrolyte is determined by this method, the analyte becomes a mobility reference probe for that background electrolyte and can be used to calculate the bulk flow mobility in subsequent conventional CE separations utilizing the same charged interacting agent. The new method can also be used to probe the interactions of the charged interacting agents and the wall of the capillary.     

Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen--antibody or oligonucleotide--DNA sensing processes by the precipitation of an insoluble product on electrodes

Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) are used as a probe to amplify the sensing of antigen-antibody interactions or oligonucleotide-DNA binding. The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H2O2, and the precipitation of the insoluble product 2 on electrode supports, are used as an amplification route for the sensing processes. The anti-dinitrophenyl antibody (DNP-Ab) is sensed by a dinitrophenyl-L-cysteine antigen monolayer associated with an Au electrode. A biotinylated anti-IgG-antibody (Fc-specific) is linked to the antigen-DNP-Ab complex, and the biotin-labeled HRP-liposomes associate with the assembly through an avidin bridge. The biocatalyzed precipitation of 2 on the electrode increases the electron-transfer resistances at the electrode-solution interface or the electrode resistance itself. The binding events of the different proteins on the electrode and the biocatalyzed precipitation of 2 on the conductive support are followed by Faradaic impedance spectroscopy or constant-current chronopotentiometry. DNP-Ab concentrations as low as 1 x 10(-11) g x mL(-1) can be detected by this method. The labeled liposomes were also used for the amplified detection of DNA 3. The oligonucleotide 4, complementary to a part of the target DNA 3 that is a model nucleic acid sequence for the Tay-Sachs genetic disorder, is assembled on an Au electrode. Hybridization of the analyte 3 followed by the association of the biotin-tagged oligonucleotide 5 yields a three-component double-stranded assembly. Sensing of the analyte 3 is amplified by the association of avidin, the labeled liposomes, and the subsequent biocatalyzed precipitation of 2 on the electrodes. The DNA 3 is detected with a sensitivity that corresponds to 6.5 x 10(-13) M. Faradaic impedance spectroscopy and chronopotentiometry were employed to follow the stepwise assembly of the systems and the electronic transduction of the detection of the analyte DNA 3.     

Detection of surface antigens on living cells through incorporation of immunorecognition into the distinct positioning of cells with positive and negative dielectrophoresis

Rapid determination of surface antigens on cells is possible by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). The DEP device for cell manipulation comprises a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO microband array electrode (band electrode) modified with an antibody. Cells with the surface antigen introduced into the channel immediately accumulated on the surface of the band electrode during p-DEP generated by the application of ac voltage between the ITO electrode and the band electrode to immobilize by the specific antibody. The removal of accumulated cells to the gap region during n-DEP was used for rapid estimation of the residual cells with a specific surface antigen. We demonstrate here that human promyelocytic leukemia cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody. The time required for the determination of the surface antigen using this compelled accumulation of cells by p-DEP and the separation of unbound cells by n-DEP is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells and thereby enhancing throughput in the clinic and in cytobiology studies.     

Enhancement in the Sensitivity of Microfluidic Enzyme-Linked Immunosorbent Assays through Analyte Preconcentration

In this Article, we describe a microfluidic enzyme-linked immunosorbent assay (ELISA) method whose sensitivity can be substantially enhanced through preconcentration of the target analyte around a semipermeable membrane. The reported preconcentration has been accomplished in our current work via electrokinetic means allowing a significant increase in the amount of captured analyte relative to nonspecific binding in the trapping/detection zone. Upon introduction of an enzyme substrate into this region, the rate of generation of the ELISA reaction product (resorufin) was observed to increase by over a factor of 200 for the sample and 2 for the corresponding blank compared to similar assays without analyte trapping. Interestingly, in spite of nonuniformities in the amount of captured analyte along the surface of our analysis channel, the measured fluorescence signal in the preconcentration zone increased linearly with time over an enzyme reaction period of 30 min and at a rate that was proportional to the analyte concentration in the bulk sample. In our current study, the reported technique has been shown to reduce the smallest detectable concentration of the tumor marker CA 19-9 and Blue Tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without analyte preconcentration. When compared to microwell based ELISAs, the reported microfluidic approach not only yielded a similar improvement in the smallest detectable analyte concentration but also reduced the sample consumption in the assay by a factor of 20 (5 μL versus 100 μL).     

Continuous flow microfluidic demixing of electrolytes by induced charge electrokinetics in structured electrode arrays

A continuous flow microfluidic demixing process is realized. It utilizes high external electrical fields that are applied over electrically floating noble metal electrodes in an otherwise straight microchannel. The process converts axial electrical potential gradients into lateral molecular selective transport via a structure oriented ensemble of numerous electrodes. While the individual electrodes locally modify the electrolyte distribution by nonlinear electrokinetic effects and concentration polarization, the directed orientation of the electrode array combines the individual polarization zones to a dedicated molecular enrichment against the generated concentration gradient. A homogeneously concentrated electrolyte can be separated into arbitrarily shaped laminae of increased and depleted concentration by the presented microfluidic demixer.     

Optofluidic concentration: plasmonic nanostructure as concentrator and sensor

The integration of fluidics and optics, as in flow-through nanohole arrays, has enabled increased transport of analytes to sensing surfaces. Limits of detection, however, are fundamentally limited by local analyte concentration. We employ the nanohole array geometry and the conducting nature of the film to actively concentrate analyte within the sensor. We achieve 180-fold enrichment of a dye, and 100-fold enrichment and simultaneous sensing of a protein in less than 1 min. The method presents opportunities for an order of magnitude increase in sensing speed and 2 orders of magnitude improvement in limit of detection.     

Characterization of electrode fouling and surface regeneration for a platinum electrode on an electrophoresis microchip

A method of electrochemically cleaning noble metal electrodes is presented and characterized for electrophoresis microchips with electrochemical detection. First, the loss of sensitivity due to electrode fouling by serotonin is characterized as a function of injection number and analyte concentration. Signal attenuation is observed to be greater at high concentrations (100 microM) and negligible at very low concentrations (approximately 1 microM). Next, an electrochemical treatment procedure is optimized to yield sensitive and reproducible amperometric detection of the highly adsorptive compounds, serotonin and histamine. Thus, the performance of the electrode is reproducibly regenerated following as much as a approximately 99% reduction in surface activity. Utilizing the optimized three-level waveform, derived from that used for pulsed amperometric detection, detection limits as low as 78 nM and 17 microM have been obtained for serotonin and histamine, respectively. In the case of serotonin, this represents the lowest detection limit for a neurotransmitter by microchip electrophoresis with amperometric detection and the first report of amperometric detection of histamine detection at an unmodified platinum electrode. Repeated use of the electrode and application of electrochemical treatment did not appear to measurably affect the noise, longevity, metal adhesion, or physical appearance of the electrode.     

On-line concentration by analyte adsorption and subsequent laser desorption in supersonic jet spectrometry

A narrow capillary, the tip of which was restricted to form a supersonic jet, was employed for sample introduction in time-of-flight mass spectrometry. The analyte was adsorbed at the tip of the capillary, due to a temperature decrease by the Joule-Thomson effect. Then, the analyte was desorbed using a pulsed laser emitting at 532 nm, and was entrained into a carrier gas. The analyte in the jet was subsequently ionized using a pulsed laser emitting at 266 nm. The duration of the analyte passing through the ionization region was 5.4 mm in length (9 micros in time), and the signal intensity was enhanced 310-fold. This technique can also improve selectivity by controlling the nozzle temperature, since volatile compounds are not trapped at the tip of the capillary and then are not concentrated in the jet. In this approach, the analyte can be injected in a pulsed mode into a vacuum without using a complicated mechanical valve even at a repetition rate of >1 kHz from the nozzle heated at a temperature of >300 degrees C.     

Controlling analyte electrochemistry in an electrospray ion source with a three-electrode emitter cell

The inherent electrochemistry occurring at the emitter electrode of an electrospray ion source was effectively controlled by incorporating a three-electrode controlled-potential electrochemical cell into the controlled-current electrospray emitter circuit. Two different basic cell designs were investigated to accomplish this control, namely, a planar flow-by working electrode and a porous flow-through working electrode design, each operated with a potentiostat floated at the electrospray high voltage. Control of the analyte electrochemistry was tested using the indole alkaloid reserpine, which is often used to test the specifications of electrospray mass spectrometry instrumentation. Reserpine was relatively easy to oxidize (E(p) = 0.73 V vs Ag/AgCl) in the acidic electrospray medium (acetonitrile/water 1:1 v/v, 5.0 mM ammonium acetate, 0.75 vol % acetic acid) and was oxidized when the conventional electrospray emitter was used at low solution flow rate. With the proper cell auxiliary electrode configuration and adjustment of the working electrode potential, it was found that reserpine oxidation could be "turned off" at flow rates as low as 2.5 microL/min as well as at flow rates as high as 30-40 microL/min. Just as important, it was also possible to "turn on" essentially 100% oxidation of reserpine in this flow rate range. The area of the auxiliary electrode along with flow rate, which affect mass transport of analytes to this electrode, were found to be critical in controlling the electrochemical reactions in the emitter cell. Such control over analyte electrochemical reactions in the emitter has been difficult or impossible to achieve with a conventional electrospray emitter. This control is paramount in obtaining experimental results free from electrochemically generated artifacts of the analyte or in exploiting electrochemical reactions involving the analyte to analytical advantage.     

Ion-partitioning membrane-based electrochemical sensors

The application of ion-partitioning membranes on proton transducers for the development of electrochemical sensors is presented. The ion-partitioning membrane incorporates two different ionophores, one selective to protons and the other to analyte cations, as well as the necessary anionic lipophilic sites. As dictated by the electroneutrality principle, when the concentration of the analyte cations in the sample increases, the analyte cations are extracted into the membrane, displacing protons of equal charge out of the membrane. The pH-sensitive gate of a CHEMFET or the surface of a glass pH electrode is used as an internal transducer for the monitoring of the membrane proton flux. The resulting signal of the pH transducer is related to the concentration of the analyte cation present in the sample. The experiments presented here indicate that the observed CHEMFET's signal is affected by the interaction of the pH-sensitive sensor element with protons released by the polymeric membrane.     

Enhanced study and control of analyte oxidation in electrospray using a thin-channel,planar electrode emitter

A thin-channel, planar electrode emitter device is described and utilized for the study and control of electrochemical oxidation of analytes at the emitter electrode in an electrospray ion source. For analytes that are not particularly susceptible to oxidation, the planar electrode device functions analytically in a manner similar to emitter systems that utilize the more common stainless steel tubular electrodes. For more easily oxidized analytes, the device provides the means to achieve near 100% oxidation efficiency or to completely eliminate analyte oxidation through simple and rapid changes in electrode material, electrode area, electrode covering, channel height above the electrode, or solution flow rate. Compared to the use of tubular electrodes, the planar electrode emitter system provides improved flexibility in altering the nature of the electrode area and material, as well as altering analyte mass transport to the electrode surface. Each of these parameters is critical in the control of electrochemical reactions and can be easily studied or exploited with this emitter electrode configuration.     

固体氧化物燃料电池Sr系钙钛矿电极B位元素成分优化规律

采用第一性原理, 对元素周期表中3~6周期52种元素作为固体氧化物燃料电池(SOFC) Sr为A位系列钙钛矿结构电极材料B位替换元素的相关结构相的结合能进行了系统计算, 据此分析了各元素对生成立方相和六方相结构稳定性影响的趋势。通过对相关体系的成分比例进行推算, 讨论了这些实验体系在稳定性趋势图中的分布规律, 进一步对上述体系的实验数据进行分析, 得到了以Mo-Fe-Co连线为中心的成分优化区域。根据相关氧离子扩散模型的计算, 结果显示该区域形成的原因与氧空位形成能、迁移能以及禁带宽度均较为适中有关。以上理论结合实验的研究为电极材料的成分优化提供了理论指导。     

Affinity capillary electrophoresis using a low-concentration additive with the consideration of relative mobilities

Most affinity studies in capillary electrophoresis assume that the analyte concentration is much smaller than the additive concentration so that the migration of the analyte has no effect on the concentration of the additive in the capillary. However, in most medium- to high-affinity interactions, the additive concentration has to be kept rather low to observe the changes in analyte mobility before saturation is reached. In this paper, a mathematical model is developed to describe the migration behavior of the analyte in a system where the complex formed becomes concentrated to levels much greater than the original concentration of the additive due to the differences in the mobilities of the analyte, additive, and complex. The analyte is flurbiprofen, the additive is transthyretin, and the stoichiometry of the reaction between the two is 1:2. This study also provides a new algorithm to determine medium- to high-affinity binding interaction constants by CE.     

Anodic stripping voltammetry coupled on-line with inductively coupled plasma mass spectrometry: optimization of a thin-layer flow cell system for analyte signal enhancement

Parameters affecting analyte signal enhancement in anodic stripping voltammetry-inductively coupled plasma mass spectrometry (ASV-ICP-MS), using a thin-layer ASV cell and microconcentric nebulization (MCN), have been examined. Silver was used as a test analyte and was deposited at a glassy carbon working electrode. The MCN allowed use of solution flow rates that were beneficial to optimum electrolytic performance of the thin-layer cell. High analyte deposition efficiencies obtained with the thin-layer cell, combined with minimal sample consumption of the MCN, allowed substantial signal enhancement (>400 times higher than continuous nebulization level) to be obtained with 2-3 mL of sample and deposition times of less than 30 min. Signal enhancement was strongly influenced by the opposing effect of flow rate on the electrolytic deposition efficiency (deposition efficiency decreases with increasing flow rate) and on the quantity of analyte delivered to the cell (analyte mass throughput increases with increasing flow rate). Excellent linearity for stripping peak heights was demonstrated for a wide range of analyte deposition times and for peak heights and peak areas (r > 0.999) over a wide concentration range (25 ng/L-20 μg/L). Precision was good (RSD typically <3% for n = 3-6) except for a high Ag blank contributed to by corrosion of the counter electrode and by Ag diffusion from the reference electrode into the cell. Details of the flow manifold and ASV cells are discussed, along with relevant performance characteristics of the MCN.