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A simple and highly sensitive technique for laser-induced fluorescence detection on multiple channels in a plastic microchip was developed, and its effectiveness was demonstrated by laser-beam ray-trace simulations and experiments. In the microchip, with refractive index nC, A channels and B channels are arrayed alternately and respectively filled with materials with refractive indexes nA for electrophoresis analysis and nB for laser-beam control. It was shown that a laser beam entering from the side of the channel array traveled straight and irradiated all A channels simultaneously and effectively because the refractive actions by the A and B channels were counterbalanced according to the condition nA < nC < nB. This technique is thus called "side-entry laser-beam zigzag irradiation". As a demonstration of the technique, when nC = 1.53, nA = 1.41, nB = 1.66, and the cross sections of both eight A channels and seven B channels were the same isosceles trapezoids with 97° base angle, laser-beam irradiation efficiency on the eight A channels by the simulations was 89% on average and coefficient of variation was 4.4%. These results are far superior to those achieved by other conventional methods such as laser-beam expansion and scanning. Furthermore, fluorescence intensity on the eight A channels determined by the experiments agreed well with that determined by the simulations. Therefore, highly sensitive and uniform fluorescence detection on eight A channels was achieved. It is also possible to fabricate the microchips at low cost by plastic-injection molding and to make a simple and compact detection system, thereby promoting actual use of the proposed side-entry laser-beam zigzag irradiation in various fields. [ABSTRACT FROM AUTHOR]

Considering the important functions of cellular Na+ and K+ together with their cooperative efforts on various biological processes, it is significant to simultaneously detect Na+ and K+ at a single-cell level. Here, we present a novel method to discriminate and quantify simultaneously Na+ and K+ in single cells using a new near-infrared fluorescent probe associated with microchip electrophoresis. The fluorescent probe selectively responds to both Na+ and K+. The microchip electrophoresis allows accurate single-cell manipulation and effective distinction of Na+ and K+. Based on the method, the concentration of Na+ and K+ in single normal and cancer cells was compared, and the variation of Na+ and K+ in single cancer cells during the early stage of apoptotic volume decrease was monitored, which would help us to better understand the critical roles of Na+ and K+ in malignant cells and apoptosis. This method has paved a new way for the research of the synergistic function of Na+ and K+ in the regulation of various biological processes at a single-cell level. [ABSTRACT FROM AUTHOR]

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE-MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE-MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE-MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE-MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE-MS analysis. [ABSTRACT FROM AUTHOR]

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both Kd and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and Kd of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium. [ABSTRACT FROM AUTHOR]

While the use of sodium dodecyl sulfate (SDS) in separation buffers allows efficient analysis of complex mixtures, its presence in the sample matrix is known to severely interfere with the mass-spectrometric characterization of analyte molecules. In this article, we report a microfluidic device that addresses this analytical challenge by enabling inline electrospray ionization mass spectrometry (ESI-MS) of low molecular weight cationic samples prepared in SDS containing matrices. The functionality of this device relies on the continuous extraction of analyte molecules into an SDS-free solvent stream based on the free-flow zone electrophoresis (FFZE) technique prior to their ESI-MS analysis. The reported extraction was accomplished in our current work in a glass channel with microelectrodes fabricated along its sidewalls to realize the desired electric field. Our experiments show that a key challenge to successfully operating such a device is to suppress the electroosmotically driven fluid circulations generated in its extraction channel that otherwise tend to vigorously mix the liquid streams flowing through this duct. A new coating medium, N-(2-triethoxysilylpropyl) formamide, recently demonstrated by our laboratory to nearly eliminate electroosmotic flow in glass microchannels was employed to address this issue. Applying this surface modifier, we were able to efficiently extract two different peptides, human angiotensin I and MRFA, individually from an SDS containing matrix using the FFZE method and detect them at concentrations down to 3.7 and 6.3 μg/mL, respectively, in samples containing as much as 10 mM SDS. Notice that in addition to greatly reducing the amount of SDS entering the MS instrument, the reported approach allows rapid solvent exchange for facilitating efficient analyte ionization desired in ESI-MS analysis. [ABSTRACT FROM AUTHOR]

Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules. [ABSTRACT FROM AUTHOR]

The transport of ionic species through a nanochannel plays important roles in fundamental research and practical applications of the nanofluidic device. Here, we demonstrated that ionic transport selectivity of a positively charged nanochannel membrane can be tuned under a phosphoric acid gradient. When phosphoric acid solution and analyte solution were connected by the positively charged nanochannel membrane, the faster-moving analyte through the positively charged nanochannel membrane was the positively charged dye (methylviologen, MV2+) instead of the negatively charged dye (1,5-naphthalene disulfonate, NDS2-). In other words, a reversed ion selectivity of the nanochannel membranes can be found. It can be explained as a result of the combination of diffusion, induced electroosmosis, and induced electrophoresis. In addition, the influencing factors of transport selectivity, including concentration of phosphoric acid, penetration time, and volume of feed solution, were also investigated. The results showed that the transport selectivity can further be tuned by adjusting these factors. As a method of tuning ionic transport selectivity by establishing phosphoric acid gradient, it will be conducive to improving the separation of ionic species. [ABSTRACT FROM AUTHOR]

Lateral flow immunoassays (LFA) are one of the most prevalent point-of-care (POC) diagnostics due to their simplicity, low cost, and robust operation. A common criticism of LFA tests is that they have poor detection limits compared to those of analytical techniques, like ELISA, which confines their application as a diagnostic tool. The low detection limit of LFA and associated long equilibration times are due to kinetically limited surface reactions that result from low target concentration. Here, we use isotachophoresis (ITP), a powerful electrokinetic preconcentration and separation technique, to focus target analytes into a thin band and transport them to the LFA capture line, resulting in a dramatic increase in the surface reaction rate and equilibrium binding. We show that ITP is able to improve the limit of detection (LoD) of LFA by 400-fold for 90 s assay time and by 160-fold for a longer 5 min time scale. ITP-enhanced LFA (ITP-LF) also shows up to 30% target extraction from 100 μL of the sample, whereas conventional LFA captures less than 1% of the target. ITP improves the LoD of LFA to the level of some lab-based immunoassays, such as ELISA, and may provide sufficient analytical sensitivity for application to a broader range of analytes and diseases that require higher sensitivity and lower detection limits. [ABSTRACT FROM AUTHOR]

Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10-5 cm2 V-1 s-1) in the nanopores and in the nanochannel with the larger cross-sectional area. [ABSTRACT FROM AUTHOR]

The article discusses the growing advances in nanofabrication techniques and fundamental transport properties in nanofluidic devices. It describes the nanofluidic structures, barriers, and critical dimensions in nanopores, nanochannels, and nanopipets. It presents the electrokinetic effects in nanofluidic devices in the aspects of conductivity, electrophoresis, and electrospray ionization.

We present an origami paper-based electrophoretic device (oPAD-Ep) that achieves rapid (∼5 min) separation of fluorescent molecules and proteins. Due to the innovative design, the required driving voltage is just ∼10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep uses multiple, thin (180 μ/layer) folded paper layers as the supporting medium for electrophoresis. This approach significantly shortens the distance between the anode and cathode, and this, in turn, accounts for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep enables convenient sample introduction by use of a slip layer as well as easy product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. We demonstrate the use of oPAD-Ep for simple separation of proteins in bovine serum, which illustrates its potential applications for point-of-care diagnostic testing. [ABSTRACT FROM AUTHOR]

Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 106 M-1. The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation. [ABSTRACT FROM AUTHOR]

The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ~6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin. [ABSTRACT FROM AUTHOR]

We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5-116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopattemed polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that die probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecularmass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. [ABSTRACT FROM AUTHOR]

Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential or field asymmetric waveform IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with the same peak width and resolving power up to ∼400 as for peptides. The present work explores the reach of this approach for larger proteins, exemplified by cytochrome c and myoglobin. Resolution similar to that for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of the present technique to proteins under ∼20 kDa. This capability may enable one to distinguish whole proteins with differing residue sequences or localizations of post-translational modifications. Small features at negative compensation voltages that markedly grow from cytochrome c to myoglobin indicate the dipole alignment of rare conformers in accord with theory, farther supporting the concept of pendular macroions in FAIMS. [ABSTRACT FROM AUTHOR]

Iodine deficiency is the most common preventable cause of intellectual disabilities in children. Global health initiatives to ensure optimum nutrition thus require continuous monitoring of population-wide iodine intake as determined by urinary excretion of iodide. Current methods to analyze urinary iodide are limited by complicated sample pretreatment, costly infrastructure, and/or poor selectivity, posing restrictions to large-scale epidemiological studies. We describe a simple yet selective method to analyze iodide in volume-restricted human urine specimens stored in biorepositories by capillary electrophoresis (CE) with UV detection. Excellent selectivity is achieved when using an acidic background electrolyte in conjunction with dynamic complexation via α-cyclodextrin in an unmodified fused-silica capillary under reversed polarity. Sample self-stacking is developed as a novel online sample preconcentration method to boost sensitivity with submicromolar detection limits for iodide (S/N ≈ 3, 0.06 `M) directly in urine. This assay also allows for simultaneous analysis of environmental iodide uptake inhibitors, including thiocyanate and nitrate. Rigorous method validation confirmed good linearity (R2 = 0.9998), dynamic range (0.20 to 4.0 μM), accuracy (average recovery of 93% at three concentration levels) and precision for reliable iodide determination in pooled urine specimens over 29 days of analysis (RSD - 11%, n = 87). [ABSTRACT FROM AUTHOR]

Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane--glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material. [ABSTRACT FROM AUTHOR]

Target DNA fragments at 10 fM concentration (approximately 6 X 10s molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR- free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic add detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nudeic add detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nudeic add probe) conjugates upon hybridization with target nudeic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nudeotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences. [ABSTRACT FROM AUTHOR]

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 µm and thicknesses of 0.4-2.4 µm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flowisoelectric focusing (μFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only. [ABSTRACT FROM AUTHOR]

In stochastic blocking electrochemistry, microparticles generate individual current steps when they adsorb on a microelectrode and decrease the current and flux of a redox mediator reacting at the surface. The amplitude of the current step informs on particle size and landing locus, while step frequency correlates with particle transport. Here, we report a new method to estimate the average arrival velocities of single rod-shaped bacteria (bacilli). The method relies on simulating the nearby threshold distance from the surface where the bacillus no longer perturbs mediator flux and the current step approaches zero. We estimated the average velocities of bacillus arrival by dividing the threshold distance over the current step duration, a parameter that here we detect for the first time and increases with bacillus length. By comparing diffusional fluctuations to bacillus average velocity, we estimated diffusion and migration contributions as a function of bacterium size. Average arrival velocities increase with bacillus length at the same time as migration intensifies and diffusion weakens. Our analysis is universal and more effective in determining transport mode contributions than the present approach of comparing theoretical and experimental step frequencies. Uncertainty in landing locus is inconsequential because the step duration used to calculate the average arrival speed already contains such information and knowing bacillus electrophoretic mobility or ζ-potential is not needed. Additionally, by simulating and assigning edge landings to the most repeated values of current steps in a recording, we obtain bacilli lengths and widths similar to scanning electron microscopy, from which we infer landing orientation.

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS. [ABSTRACT FROM AUTHOR]

The common selenium oxoanions selenite (SeO32-) and selenate (SeO42-) are toxic at intake levels slightly below 1 mg day-1. These anions are currently monitored by a variety of traditional analytical techniques that are time-consuming, expensive, require large sample volumes, and/or lack portability. To address the need for a fast and inexpensive analysis of selenium oxoanions, we present the first microchip capillary zone electrophoresis (MCE) separation targeting these species in the presence of chloride, sulfate, nitrate, nitrite, chlorate, sulfamate, methanesulfonate, and fluoride, which can be simultaneously monitored. The chemistry was designed to give high selectivity in nonideal matrices. Interference from common weak acids is avoided by operating near pH 4. Separation resolution from chloride was enhanced to improve tolerance of high-salinity matrices. As a result, selenate can be quantified in the presence of up to 1.5 mM NaCl, and selenite analysis is even more robust against chloride. Using contact conductivity detection, detection limits for samples with conductivity equal to the background electrolyte are 53 nM (4.2 ppb Se) and 380 nM (30 ppb) for selenate and selenite, respectively. Analysis time, including injection, is ~2 min. The MCE method was validated against ion chromatography (IC) using spiked samples of dilute BBL broth and slightly outperformed the IC in accuracy while requiring <10% of the analysis time. The applicability of the technique to real samples was shown by monitoring the consumption of selenite by bacteria incubated in LB broth. [ABSTRACT FROM AUTHOR]

We present the development, formulation, validation, and demonstration of a fast, generic, and open source simulation tool, which integrates nonlinear electromigration with multispecies nonequilibrium kinetic reactions. The code is particularly useful for the design and optimization of new electrophoresis-based bioanlaytical assays, in which electrophoretic transport, separation, or focusing control analyte spatial concentration and subsequent reactions. By decoupling the kinetics solver from the electric field solver, we demonstrate an order of magnitude improvement in total simulation time for a series of 100 reaction simulations using a shared background electric field. The code can efficiently handle complex electrophoretic setups coupling sharp electric field gradients with bulk reactions, surface reactions, and competing reactions. For example, we demonstrate the use of the code for investigating accelerated reactions using isotachophoresis (ITP), revealing new regimes of operation which in turn enable significant improvement of the signal-to-noise ratio of ITP-based genotypic assays. The user can define arbitrary initial conditions and reaction rules, and we believe it will be a valuable tool for the design of novel bioanalytical assays. We will offer the code as open source, and it will be available for free download at http://microfluidics.technion.ac.il. [ABSTRACT FROM AUTHOR]

Small-volume continuous-flow synthesis (small-vol- Conventional Device Optimized Device nme CFS) offers a number of benefits for use in small-scale chemical production and exploratory chemistry. Typically, small-volume CFS is followed by discontinuous purification; however, a fully continuous synthesis-purification combination is more attractive. Milli free-flow electrophoresis (mFFE) is a promising continuous-flow purification technique that is well suited for integration with small-volume CFS. The purification stability of mFFE, however, needs to be significantly improved before it can be feasible for this combination. One of the major sources of instability of mFFE is attributed to the ions produced as a result of electrolysis. These ions can form pH and conductivity gradients in mFFE, which are detrimental to separation quality. The severity of these gradients has not been thoroughly studied in mFFE. In this paper, we have experimentally demonstrated that detrimental pH gradients occur at flow rates of 8 mL/ min and less, and electric field strengths of 25 V/cm and greater. To decrease the pH gradients, it is necessary to evacuate H+ and OH- as soon as they are generated; this can be done by increasing local hydrodynamic flow rates. We calculated the necessary flow rate, to be applied at the electrode, which can effectively wash away both ions before they can cause a detrimental pH gradient These optimized flow rates can be attained by designing a device that incorporates deep channels. We have confirmed the effectiveness of these channels using a prototyped device. The new design allows mFFE users to work over a wider range of flow-rate and electric-field conditions without experiencing significant changes in pH. [ABSTRACT FROM AUTHOR]

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in μFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC X μFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC X μFFE peak capacities as high as 2.3S2 could be obtained in a 10 min separation window when determined according to die product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC X μFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC X LC. [ABSTRACT FROM AUTHOR]

Mitochondrial membrane potential varies, depending on energy demand, subcellular location, and morphology and is commonly used as an indicator of mitochondrial functional status. Electrophoretic mobility is a heterogeneous surface property reflective of mitochondrial surface composition and morphology, which could be used as a basis for separation of mitochondrial subpopulations. Since these properties are heterogeneous, methods for their characterization in individual mitochondria are needed to better design and understand electrophoretic separations of subpopulations of mitochondria, Here we report on the first method for simultaneous determination of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis witii laser-induced fluorescence detection (CE-LIF). Mitochondria were isolated from cultured cells, mouse muscle, or liver, and then polarized, labeled with JC-1 (a ratiometric fluorescent probe, which indicates changes in membrane potential), and separated with CE-LIF. Red/green fluorescence intensity ratios from individual mitochondria were used as an indicator of mitochondrial membrane potential. Reproducible distributions of individual mitochondrial membrane potential and electrophoretic mobility were observed. Analysis of polarized and depolarized regions of interest defined using red/green ratios and runs of depolarized controls allowed for the determination of membrane potential and comparison of electrophoretic mobility distributions in preparations containing depolarized mitochondria. Through comparison of these regions of interest, we observed dependence of electrophoretic mobility on membrane potential, with polarized regions of interest displaying decreased electrophoretic mobility. This method could be applied to investigate mitochondrial heterogeneity in aging or disease models where membrane potential is an important factor. [ABSTRACT FROM AUTHOR]

Isotachophoresis (ITP) and electric field grad ient focusing (EFGF) are two powerful approaches for simultaneous focusing and separation of charged compounds. Remarkably, in many EFGF methods, isotachophoretic hallmarks have been found, including observations of plateau concentrations and contiguous analyte bands. We discuss the similarities between ITP and EFGF and describe promising possibilities to transfer the functionality and applications developed on one platform to other platforms. Of particular importance is the observation that single-electrolyte isotachophoretic separations with tunable ionic mobility window can be performed, as is illustrated with the example of depletion zone isotachophoresis (dzITP). By exploiting the rapid developments in micro- and nanofluidics, many interesting combinations of ITP and EFGF features can be achieved, yielding powerful analytical platforms for sample preparation, biomarker discovery, molecular interaction assays, drug screening, and clinical diagnostics. [ABSTRACT FROM AUTHOR]

We describe a chemical vapor deposition (CVD) method for the surface modification of glass microfluidic devices designed to perform electrophoretic separations of cationic species. The microfluidic channel surfaces were modified using aminopropyl silane reagents. Coating homogeneity was inferred by precise measurement of the separation efficiency and electroosmotic mobility for multiple microfluidic devices. Devices coated with (3-aminopropyl)di-isopropylethoxysilane (APDIPES) yielded near diffusion-limited separations and exhibited little change in electroosmotic mobility between pH 2.8 and pH 7.5. We further evaluated the temporal stability of both APDIPES and (3-aminopropyl)trietiioxysilane (APTES) coatings when stored for a total of 1 week under vacuum at 4 °C or filled with pH 2.8 background electrolyte at room temperature. Measurements of electroosmotic flow (EOF) and separation efficiency during this time confirmed that both coatings were stable under both conditions. Microfluidic devices with a 23 cm long, serpentine electrophoretic separation channel and integrated nanoelectrospray ionization emitter were CVD coated with APDIPES and used for capillary electrophoresis (CE)-electrospray ionization (ESl)-mass spectrometry (MS) of peptides and proteins. Peptide separations were fast and highly efficient, yielding theoretical plate counts over 600,000 and a peak capacity of 64 in less than 90 s. Intact protein separations using these devices yielded Gaussian peak profiles with separation efficiencies between 100,000 and 400,000 theoretical plates. [ABSTRACT FROM AUTHOR]

A dual-channel sequential injection microchip capillary electrophoresis system with pressure-driven injection is demonstrated for simultaneous separations of anions and cations from a single sample. The poly(methyl methacrylate) (PMMA) microchips feature integral in-plane contactless conductivity detection electrodes. A novel, hydrodynamic "split-injection" method utilizes background electrolyte (BGE) sheathing to gate the sample flows, while control over the injection volume is achieved by balancing hydrodynamic resistances using external hydrodynamic resistors. Injection is realized by a unique flow-through interface, allowing for automated, continuous sampling for sequential injection analysis by microchip electrophoresis. The developed system was very robust, with individual microchips used for up to 2000 analyses with lifetimes limited by irreversible blockages of the microchannels. The unique dual-channel irreversible blockages of the microchannels. The unique dual-channel geometry was demonstrated by the simultaneous separation of three cations and three anions in individual microchannels in under 40 s with limits of detection (LODs) ranging from 1.5 to 24 μM. From a series of 100 sequential injections the %RSDs were determined for every fifth run, resulting in %RSDs for migration times that ranged from 0.3 to 0.7 (n = 20) and 2.3 to 4.5 for peak area (n = 20). This system offers low LODs and a high degree of reproducibility and robustness while the hydrodynamic injection eliminates electrokinetic bias during injection, making it attractive for a wide range of rapid, sensitive, and quantitative online analytical applications. [ABSTRACT FROM AUTHOR]

Detecting possible modifications of therapeutic proteins is a critical element of the quality control of protein drugs. Typically, a number of techniques are used to evaluate different modifications of therapeutic protein formulations. Using heteronuclear NMR spectroscopy, we show that the difference between various insulin formulations can be detected "as is" with little pretreatment and quickly. As an application to the quality control of insulin formulations, the NMR approach was compared with four different analytical methods: with reverse phase high pressure liquid chromatography (HPLC) (for mutations), with size exclusion chromatography (for oligomerization), with electrophoresis (for denaturation), and with mass spectrometry (for deamidation). All of the results showed that this single NMR method can provide the specific signatures for each modification and information that is at least equivalent to that offered by the conventional analytical methods. Importantly, NMR could yield information at each amino acid residue level which no other technique provided. The suggested NMR method, then, can be considered to be a facile and effective means of evaluating therapeutic protein formulations in a multifaceted way. [ABSTRACT FROM AUTHOR]

We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min-1 mg-1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min-1 mg-1 and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min-1 mg-1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min-1 mg-1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. [ABSTRACT FROM AUTHOR]

Here, we report on DNA electrophoresis through a novel artificial sieving matrix based on the highly regular submicrometer cylindrical glass capillary segments alternatingly arranged with wells. Such round capillaries pose a higher-order confinement resulting in a lower partition coefficient and greater entropic energy barrier while limiting the driving field strength to a small fraction of the applied electric field. In return, the separation can be performed at high average field strengths (up to 1.6 kV/cm) without encountering the field-dependent loss of resolving power. This leads to fast DNA sieving as demonstrated here on the capillaries of 750 nm in diameter. The 600 bp to 21 kbp long chains are shown to resolve within 4 min after having undergone a fairly limited number of entropic barriers (128 in total). The capillary matrix also exhibits a critical field threshold below which DNA bands fail to launch, and this occurs at a considerably greater magnitude than in other matrixes. The submicrometer capillaries are batch-fabricated on silicon through a fabrication process that does not require high-resolution advanced lithography or well-controlled wafer bonding techniques to define their critical dimension. [ABSTRACT FROM AUTHOR]

In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3--6 μm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a gfven pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be mininuzed by using ion selectivity of hydVogel-iniiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity. [ABSTRACT FROM AUTHOR]