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We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 min of continuous signal enhancement, we can detect 4.4 pM human immunoglobulin E (IgE) and 9 pM human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT), which are among the lowest limits of detection (LOD) reported. IgE was detected in serum sample with a LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays. [ABSTRACT FROM AUTHOR]

Single-cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in the positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15) chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples. [ABSTRACT FROM AUTHOR]

Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components—for example, cells, proteins, or particulates in complex samples that can interfere with analysis—from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples. [ABSTRACT FROM AUTHOR]

A strategy based on postcolumn electrophoretic mobility control (EMC) was developed to alleviate the adverse effect of trifluoroacetic acid (TFA) on the liquid chromatography-mass spectrometry (LC-MS) analysis of peptides. The device created to achieve this goal consisted of a poly(dimethylsiloxane) (PDMS)-based junction reservoir, a short connecting capillary, and an electrospray ionization (ESI) sprayer connected to the outlet of the high-performance liquid chromatography (HPLC) column. By apply different voltages to the junction reservoir and the ESI emitter, an electric field was created across the connecting capillary. Due to the electric field, positively charged peptides migrated toward the ESI sprayer, whereas TFA anions remained in the junction reservoir and were removed from the ionization process. Because TFA did not enter the ESI source, ion suppression from TFA was alleviated. Operation of the postcolumn device was optimized using a peptide standard mixture. Under optimized conditions, signals for the peptides were enhanced 9-35-fold without a compromise in separation efficiency. The optimized conditions were also applied to the LC-MS analysis of a tryptic digest of bovine serum albumin. [ABSTRACT FROM AUTHOR]

We present a novel technique for coupling isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis (ITP). Bidirectional ITP simultaneously sets up sharp ITP interfaces between relatively high- and low-mobility cations and high- and low-mobility anions. These two interfaces can migrate toward each other and be described as ion concentration shock waves. We here demonstrate a bidirectional ITP process in which we use the interaction of these anionic and cationic ITP shock waves to trigger a transformation from ITP preconcentration to electrophoretic separation. We use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. The method provides faster and much less dispersive transition from ITP preconcentration to electrophoretic separation compared with traditional (unidirectional) transient ITP. It eliminates the need for intermediate steps between focusing and separation, such as manual buffer exchanges. We illustrate the technique with numerical simulations of species transport equations. We have validated our simulations with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high-resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP. [ABSTRACT FROM AUTHOR]

Segmented flow in microfluidic devices involves the use of droplets that are generated either on- or off-chip. When used with off-chip sampling methods, segmented flow has been shown to prevent analyte dispersion and improve temporal resolution by periodically surrounding an aqueous flow stream with an immiscible carrier phase as it is transferred to the microchip. To analyze the droplets by methods such as electrochemistry or electrophoresis, a method to "desegment" the flow into separate aqueous and immiscible carrier phase streams is needed. In this paper, a simple and straightforward approach for this desegmentation process was developed by first creating an air/water junction in natively hydrophobic and perpendicular PDMS channels. The air-filled channel was treated with a corona discharge electrode to create a hydrophilic/hydrophobic interface. When a segmented flow stream encounters this interface, only the aqueous sample phase enters the hydrophilic channel, where it can be subsequently analyzed by electrochemistry or microchip-based electrophoresis with electrochemical detection. It is shown that the desegmentation process does not significantly degrade the temporal resolution of the system, with rise times as low as 12 s reported after droplets are recombined into a continuous flow stream. This approach demonstrates significant advantages over previous studies in that the treatment process takes only a few minutes, fabrication is relatively simple, and reversible sealing of the microchip is possible. This work should enable future studies in which off-chip processes such as microdialysis can be integrated with segmented flow and electrochemical-based detection. [ABSTRACT FROM AUTHOR]

A simple, rapid, and highly effective technique for concentrating charged macromolecules is described which employs electrophoresis in a conic cell made of a dialysis membrane. The cell is partly submerged in electrolyte solution, and the level of solution slowly moves down during the process. The electric field within the cell is at its maximum in the area that is level with the surface of the external solution. This maximum value increases and its location moves downward following the decreasing level of external solution carrying downward and concentrating charged macromolecules. It has been demonstrated that proteins can be concentrated within 12-15 min by a factor of ~100 000 with the total yield of 60-80%. Concentrated proteins can be harvested from the nanoliter-sized cul-de-sac of the conic concentrator using chemically activated magnetic beads. The presence of certain protein molecules linked to the bead's surface can be further revealed by specific reaction with a microarray of antibody molecules. Such "reversed magnetic array" format was applied to a cone-concentrated exhaled breath condensate (EBC) to reveal the presence of human immunoglobulin in the EBC and to estimate its concentration. The technique may be used for concentrating and detecting trace amounts of pathogens and toxins, in protein crystallization, and in many other applications. [ABSTRACT FROM AUTHOR]

Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like micro- dialysis. Because of the low flow rates used (50 nL/min), it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/mm by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked L-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3-4 injections each) and rise times of 22 ± 2s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments. [ABSTRACT FROM AUTHOR]

A repeatable preconcentration electrophoretic methodology for the analysis of bacteria was developed. This method is based on an isotachophoretic mode coupled with a simultaneous hydrodynamic-electrokinetic injection in conditions of field-amplified sample injection. This electrophoretic method allows the quantification of Enterobacter cloacae (studied as a model of Gram negative bacteria) with a limit of detection of 2 × 104 cells/mL. With the optimized conditions, a preconcentration factor of about 500-fold was obtained as compared to a standard hydrodynamic injection. The RSD (n = 5) on the migration time and on the peak area were 3% and 5%, respectively. This capillary electrophoretic methodology has been applied for the quantification of microbes in natural water (river and natural spring waters). Filtration of the sample prior to injection was required to remove ions present in the water and to keep the field-amplified sample injection condition at the injection. Filtrated bacteria were then recovered in terminating electrolyte diluted 10 times with water. Good agreements were obtained between cellular ATP measurements and the proposed CE methodology for the quantification of bacteria in waters. [ABSTRACT FROM AUTHOR]

When coupling capillary electrophoresis with postcolumn detection methods, such as mass spectrometry, the presence of postcolumn band broadening must be considered. The band broadening effects introduced by junction-at-the-tip CE-MS interfaces using a postcolumn micro flow-through vial are investigated by studying the hydrodynamic flow patterns and mass transport process inside the micro vial at the end of the CE separation capillary. Simulation results obtained by solving the Navier-Stokes and mass balance equations provide insights into the velocity field and concentration distribution of the analytes in the micro vial and demonstrate that, with a low flow rate of chemical modifier solution, the laminar flow streams confine the analyte molecules to the central part of the micro vial and thus maintain major features of the peak shapes. Peaks detected by UV and MS under similar experimental conditions were compared to verify the numerical prediction that the main features of the UV peak can be retained in the MS peak. Experiments also show that band broadening can be minimized when an appropriate chemical modifier flow rate is selected. [ABSTRACT FROM AUTHOR]

The article examines the methodology for evaluating quality, purity, strength, and stability of pharmaceuticals and related drugs. It highlights the applications of mass spectrometry in combination with high performance liquid chromatography to profile complex mixtures of medical compounds found in herbal. It reviews the separation-based techniques that deal the routine stability testing of liquid chromatography and chiral analysis of pharmaceuticals by capillary electrophoresis.

It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to eight samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to eight samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/rnL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid, and high-throughput analysis. [ABSTRACT FROM AUTHOR]

In this work, the utilization of matrix-assisted laser desorption/ionizjtion-mass spectrometric imaging (MALDl-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves slidingthe CE capillar)- distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MAl.PI-MSi by-time of flight (TOFJ/TOF along the CE track enables high-resolution and high-sensitivity detection of'peptides, allowing the reconstruction of a CE electro pherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide Standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDl-MS direct analysis and the theoretical ratios. The CE- MALDl-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range. [ABSTRACT FROM AUTHOR]

Label-tree oligonucleotide sensors that use a change in the electrode kinetics of the redox reaction of the negatively charged Fe(CN)63-/4- redox couple to signal the formation of a DNA duplex with a surface-conjugated probe nucleotide are investigated. Electrochemically active conducting polymers (ECPs) can advantageously be used both as the active electrode and as the means of surface conjugation of the probe nucleotide. Here, we demonstrate that the sensitivity of the detection of the surface-complementary oligonucleotide can significantly be improved, into the low nanomolar range, by forming the ECP as a highly porous, very rough layer by growing it using electrochemical polymerization on a microelectrode. In comparison, smoother surtaces formed on macroelectrodes had detection sensitivity in the low micromolar range. We propose Donnan exclusion of the redox couple from small pores as the reason for the enhanced sensitivity. We discuss the effects using a simple patch model for the electrochemical kinetics and use the model to derive the equilibrium binding constant and binding kinetic rate constants for the surface hybridization reaction. We use the electro chemically active copolymer of pyrrole (Py) and 3-pyrrolylacrylic acid (PAA) [poly(Py-co-PAA)] as the sensing electrode and binding surface and measure the surface hybridization-induced changes in electrode kinetics of Fe(CN)63- /4- by electrochemical impedance spectroscopy. [ABSTRACT FROM AUTHOR]

A mass spectrometry (MS)-compatible, isoelectric point-based separation method for removal of neutral additives from protein solutions is described. The separation is performed by electrophoretic migration and trapping using a device referred to as membrane separated wells for isoelectric focusing and trapping (MSWIFT). Electrophoretic separation in the MSWIFT device is fast; the entire process can be carried out in a matter of minutes, and it does not require further sample cleanup prior to MS analysis. Proof-of-concept experiments in which neutral additives (e.g., Triton X-100, Tween 20, poly(ethylene glycol)) are removed from protein solutions using the MSWIFT device followed by MS analysis are described. Coupling the MSWIFT separation with ion mobility MS provides additional separation via the gas phase and assists in achieving higher quality ESI mass spectra when small amounts of additives remain in solution. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480000 to 640000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes α1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins α1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells. [ABSTRACT FROM AUTHOR]

Drosophila, the fruit fly, is a common model organism in biology; however, quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry (CE-FSCV) detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w118 flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), the serotonin tissue content was greater than for Ddc-GAL4 but not significantly different than the wild-type. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva. [ABSTRACT FROM AUTHOR]

A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)) was essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a polyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another. [ABSTRACT FROM AUTHOR]

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ~1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot. [ABSTRACT FROM AUTHOR]

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe anda 1.8 T permanent magnet to measure 19F NMIR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system. [ABSTRACT FROM AUTHOR]

A novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting two- dimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri and mouse blood samples. First, a new sweeping with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhance detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely high resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug lengths for flavonoids under 15 kV for 60 mm were experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8% (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3 × 104 plates/m. This method has been successfully applied to determine fiavonoids in Herba Leonuri and postdosing mouse blood samples. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples. [ABSTRACT FROM AUTHOR]

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases. [ABSTRACT FROM AUTHOR]

Salts and buffers, commonly used in isolation and stabilization of biological analytes, have a deleterious effect on electrospray ionization mass spectrometry (ESI-MS). Excessive concentrations of salts lead to ion suppression and adduct formation, which mask or complicate ion signals. In this work, we describe a salt remover (SR), configured as a three-compartment flow-through system, where the central compartment is separated from the outer compartments by a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM). One platinum electrode is placed in each of the outer compartments, where water or electrolyte is flowing. The CEM electrode is held at a negative potential relative to the AEM side; cations/anions migrate by electrophoresis to the CEM/AEM receiver liquids, respectively. Proteins have poorer electrophoretic mobility relative to the buffer components, permitting removal of the salt. The salt-free proteins proceed to the ESI source. The capillary scale SR (intemal volume 2.5 μL) described here effectively desalted continuous flows of NaCl solutions (200 mequiv/L at 1 μL/min, equivalent to a flux of 200 nequiv/min with 88% efficiency) and achieved >99.8% salt removal with 154 mM NaCl (isotonic saline) at 1 μL/min. With optimized current, >80% of concurrently present 20uM protein was transmitted. Desalting efficiency and analyte loss was evaluated with different salt concentration and flow rate combinations under different applied current. Good-quality ESI-MS spectra of cytochrome c, myoglobin, and lysozyine as test proteins in a saline solution, passed through the SR, are demonstrated. [ABSTRACT FROM AUTHOR]

This technical note describes a new chemiluminescence immunoassay hyphenated to capillary electrophoresis (CE-based CL-IA) with gold nanoparticles (AuNPs) technique for biological molecules determination. AuNPs were used as a protein label reagent in the light of its excellent catalytic effect to the CL reaction of luminol and hydrogen peroxide. AuNPs conjugate with antibody (Ab) to form tagged antibody (Ab*), and then Ab* link to antigen (Ag) to produce an Ab*-Ag complex by a noncompetitive immunoreaction. The mixture of the excess Aba and the Aba*-Ag complex was baseline separated and detected within 5 mm under the optimized conditions. This new protocol was evaluated with human immunoglobulin G (lgG) as the target molecule. The calibration curve of IgG was in the range of 0.008-5 μg/mL with a correlation coefficient of 0.995. The detection limit (S/N = 3) of lgG was 1.14 × 10-3 μg/mL (7.1 pmol/L, 0.39 amol). The proposed AuNPs enhanced CE- based CL-IA method was successfully applied for the quantification of IgG in human sera from patients. It proves that the present method could be developed into a new and sensitive biochemical analysis technique. [ABSTRACT FROM AUTHOR]

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selec- tively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers. [ABSTRACT FROM AUTHOR]

Elecfrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of "drag-tag" as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+ 1 to +6) and yet highly water-soluble. Using a four-color UF detector, 265 bases could be read in 30 mm with a 267-amino acid drag-tag, on par with the average read of current next-gen sequenctog systems. New tWes of multichannel systems that allow much higher thmughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix. [ABSTRACT FROM AUTHOR]

Ion mobility spectromeuy (IMS), and particulaily differential IMS or field asyinmetiic waveform IMS (FAIMS), is emerging as a versatile tool for separation and identification of gasphase ions, especially in conjunction with mass spectromeli)'. For over two decades since its inception, the utility of FAIMS was conslmined by resolving power (R) of less than ∼20. Stronger electric fields and optimized gas mixtures have recently raised achievable R to ∼200, but further progress with such approaches is impeded by electrical breakdown. However, the resolving power of planar FAIMS devices using any gas and field intensity scales as the square root of separation time (0. Here, we extended t from the previous maximum of 0.2 s up to 4-fold by reducing the carrier gas flow and increased the resolving power by up to 2-fold as predicted, to >300 for multiply charged peptides. The resulting resolution gain has enabled separation of previously 'co-eluting" peptide isomers, including folding conformers and localization variants of modified peplides. More broadly, a peak capacity of ∼200 has been reached in tryplic digest separations. [ABSTRACT FROM AUTHOR]

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 μm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 μm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome. [ABSTRACT FROM AUTHOR]

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shildmate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. in positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18]+ or [M + 23]+, after protonated molecule detection (m/z = [M + 1]+) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = EM - it), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism. [ABSTRACT FROM AUTHOR]

The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an Important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycospbingolipids in infected red blood cells (IRBCs). Atetramethyirhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillaiy electrophoresis with laser-induced fluorescence detection. The lysate from eiythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique. [ABSTRACT FROM AUTHOR]

The sensitivity of enantioselective cyclodextrin-induced mom-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with α-cyclodexlrin (α-CD). The coffisional Dexter energy transfer fmm the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is sill veiy efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (±)-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20mM α-CD, 10mM cathoxymethyl-β-CD, and 25mM borate buffer at pH 9.0. `lime-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)and (-)-CQ in the presence of both α-CD and CM-β-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 mn with a pulsed, small-sized, quadrupled Nd: YAG laser. With 1 x 10-5 M 2,6-NS, limits of detection of 4.1 x 10-8M and 5.2 x 10-8Mwerefoundfor(+)CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ± 8 ps for (+)-CQ and 126 ± 10 ps for (-)-CQ. The method was used to detennine the concentration of (±)-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ± 0.1 x 10-7 M of both (+)-CQ and (-)-CQ. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are intnduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a chargecoupled device. Characterization of the system shows that injection volume is conlaniled by sample plugvolume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 mm was achieved. Further development of the system may allow application to highthroughput applications such as drug screening. [ABSTRACT FROM AUTHOR]

Nucleic acid based affinity reagents (e.g., aptalners) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electmphoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift eledrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-κB using a thiomodified aptamer) in an integrated electrophoretic gelshift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target. [ABSTRACT FROM AUTHOR]

Joule heating always accompanies electrophoresis and unavoidably leads to a temperature increase of the electrolyte. The elevated temperatures are known to adversely affect the quality of separation and detection. To minimize the temperature increase in capillary electrophoresis (CE), Joule heat is removed by actively cooling the capillary. However, there are always small parts of the capillary, such as its inlet, outlet, and detection window, which are not actively cooled. The noncooled capillary inlet has been recently proven to have an elevated temperature which is high enough to significantly affect CE-based quantitative affinity analyses. The temperature difference between the cooled and noncooled regions has never been determined due to the lack of a suitable method. Here, we report on the first experimental determination of temperature in the cooled part of the capillary and the noncooled inlet region of the capillary. We found that, under typical CE conditions, with a low-conductivity run buffer, the temperature in the noncooled inlet exceeded the temperature in the cooled region by more than 15 °C. High-conductivity buffers are anticipated to have even greater temperature differences between the noncooled and cooled capillary parts. Our results strongly suggest the potential effect of the noncooled capillary regions on the quality of CE-based analyses, which cannot be ignored. The simplest way to avoid potential errors is to move the sample to the cooled region by pressure or by applying a low electric field. [ABSTRACT FROM AUTHOR]

Electro-migration techniques, such as electrophoresis, are widely utilized in analytical sciences. If a single electrolyte is used, the field strength is typically assumed to be well-defined. Heat-associated field distortion (HAFD) has been suggested as a result of the nonuniform heat dissipation throughout the electrolyte; however, it has never been experimentally studied. Here, we experimentally demonstrated HAFD for the first time. We used capillary electrophoresis (CE) with a capillary having parts with different heat dissipation efficiencies. Our experiments showed a difference in field strength of approximately 1.5 times between the different parts of the capillary for a typical CE electrolyte. This result suggests that HAFD is a well pronounced phenomenon that can be a potential source of errors and instabilities in electro-migration experiments. [ABSTRACT FROM AUTHOR]

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1 x phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/ml were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases. [ABSTRACT FROM AUTHOR]

Permeabilization of model lipid membranes by virus-derived peptides, viral proteins, and entire virions of human rhinovirus was assessed by quantifying the release of a fluorescent dye from liposomes via a novel chip electrophoretic assay. Liposomal leakage readily occurred upon incubation with the pH-sensitive synthetic fusogenic peptide GALA and, less efficiently, with a 24mer peptide (P1-N) derived from the N-terminus of the capsid protein VP1 of human rhinovirus 2 (HRV2) at acidic pH. Negative stain transmission electron microscopy showed that liposomes incubated with the rhinovirus-derived peptide remained largely intact. At similar concentrations, the GALA peptide caused gross morphologtcal changes of the liposomes. On a molar basis, the leakage-inducing efficiency of the P1 peptide was by about 2 orders of magnitude inferior to that of recombinant VP1 (from HRV89) and entire HRV2. This underscores the role in membrane destabilization of VP1 domains remote from the N-terminus and the arrangement of the peptide in the context of the icosahedral virion. Our method is rapid, requires tiny amounts of sample, and allows for the parallel determination of released and retained liposomal cargo. [ABSTRACT FROM AUTHOR]

Understanding nanoparticle-protein interaction could help in promoting applications of nanoparticles in the biomedical fields and reducing/preventing possible adverse effects to the biological systems caused by nanoparticles. Quantitative measurement of the biophysical parameters of nanoparticle-protein interaction will improve such understanding, which could be conveniently performed by capillary electrophoresis (CE) as demonstrated in the present study. Two interaction situations were identified. Stable nanoparticle-protein complexes were resolved from the free nanoparticles and the proteins by capillary zone electrophoresis (CZE). Transient complexes with fast association/dissociation rates showed distinct mobility change from the free nanoparticles in affinity capillary electrophoresis (ACE). Interactions of bovine serum albumin (BSA) with the Fe3O4 nanoparticles (average diameters of 8 and 10 nm) and with the Au nanoparticles (average diameters of 5 and 10 nm) displayed slow and fast binding kinetics, respectively. Using the Hill equation, we could calculate the dissociation constants (KD) and cooperativity coefficients (n). Impacts on nanoparticle-protein interaction from the physicochemical properties of nanoparticles and the incubation buffer were evaluated on the basis of the KD and n values to interpret the interaction driving forces. Our study demonstrated the high simplicity and flexibility of CE in probing the interaction of proteins with diverse particles. The separation power of CE should also facilitate studies of the multicomponent interaction systems for investigating how adsorption onto nanoparticles could affect the protein-protein or protein-small molecule interactions. [ABSTRACT FROM AUTHOR]

Two synthetic schemes to produce a hydride-modified support that serves as an intermediate for the preparation of bonded phases for liquid chromatography (LC) and capillary electrophoresis (CE) are investigated. The strategies differ in the silane reagent utilized (trichlorosilane (TCS) or triethoxysilane (TES)) and the manner water is incorporated into the reaction. In the first approach, TCS in toluene reacts with a previously humidified silica substrate so that the reaction is confined to the silica surface. In the second approach, TES and a small amount of aqueous HCl are dissolved in THF, and this hydrolysate is diluted by a great factor in cyclohexane, prior to reaction with the silica substrate. Atomic force microscopy (AFM) images of the hydride film on wafers revealed that, unlike the traditional approach that produced a patchy coating, both new methods provided a homogeneous layer on the substrate's surface. IR and NMR spectra from porous silica particles clearly confirmed a successful surface modification. AFM and water contact angles (WCA) were used to examine the effect of dilution of the TES hydrolysate in cyclohexane on the trend of the film to polymerize on wafers and found that a dilution factor of at least 100 is required to attain a molecularly thin hydride layer. WCA and CE also revealed a strong susceptibility of the hydride silica intermediate to hydrolyze, even at low pH. Compared to TCS, the lower reactivity and volatility of TES resulted in a much more desirable experimental approach. [ABSTRACT FROM AUTHOR]

The study of interactions between oppositely multicharged (macro)molecules remains a challenging issue. In frontal analysis capillary electrophoresis (FACE), it is difficult to avoid the adsorption of one of the interacting partners onto the capillary wall. In this work, we demonstrate the possibility to use FACE and affinity capillary electrophoresis (ACE) on a neutrally coated capillary for the study of interactions between a polycationic dendrigraft (or linear) poly-l-lysines, on one hand, and a multicharged anionic biomolecule (adenosine monophosphate, AMP, or adenosine triphosphate, ATP), on the other hand. A systematic comparison of four different neutral coatings (hydroxypropyl cellulose, polydimethylacrylamide, polyacrylamide, polyethylene glycol) has been performed based on the repeatability of the electrophoretic migration of the dendrigraft poly-l-lysines at pH close to neutrality. Both FACE and ACE methodogies were then used to study the interactions and to get the association constants and the stoichiometry of the complex. Multisite interactions, with two classes of independent sites, were determined. The specificity of the dendritic polylysine structure compared with linear polylysine in the interaction with ATP or AMP is also emphasized. [ABSTRACT FROM AUTHOR]

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h. [ABSTRACT FROM AUTHOR]

Field-amplified sample stacking using a fused silica caplllary coated with gold nanoparticles (AuNPs) embedded in poly(diallyl dimethylammonium) chloride (PDDA) has been investigated for the electrophoretic separation of indoxyl sulfate, homovanillic acid (HVA), and vanillylmandelic acid (VMA). AuNPs (27 nm) exhibit ionic and hydrophobic interactions, as well as hydrogen bonding with the PDDA network to fonn a stable layer on the internal wall of the capillary. This approach reverses electro-osmotic flow allowing for fast migration of the analytes while retarding other endogenous compounds including ascorbic acid, uric acid, catecholamines, and indoleamines. Notably, the two closely related biomarkers of clinical significance, HVA and VMA, displayed differential interaction with PDDA-AuNPs which enabled the separation of this pair. The detection limit of the three analytes obtained by using a boron doped diamond electrode was ∼75 nM, which was significantly below their nonnal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfering chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. [ABSTRACT FROM AUTHOR]

The applicability of an online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP), to microchip zone electrophoresis (MCZE) for the analysis of oligosaccharides was investigated. Since the sample stacking and separation proceeded continuously without polarity switching in LVSEP, a single "straight" channel microchip could be employed. In the MCZE analysis of oligosaccharides, sample adsorption onto the channel surface should be suppressed, so the straight microchannel was modified with poly(vinyl alcohol) (PVA). So far, the mechanism of LVSEP in the polymer-coated capillary or microchannel has not been reported, and thus, the LVSEP process in the PVA-coated channel was investigated by fluorescence imaging. Although it is well-known that the PVA coating can suppress the electroosmotic flow (EOF), an enhanced EOF with a mobility of 4.4 x 10-4 cm²/(V·s) was observed in a low ionic strength sample solution. It was revealed that such temporarily enhanced EOF in the sample zone worked as the driving force to remove the sample matrix in LVSEP. To evaluate the analytical performance of LVSEP-MCZE, oligosaccharides were analyzed in the PVA-coated straight channel. As a result, both the glucose ladder and oligosaccharides obtained from bovine ribonuclease B were well enriched and separated with up to 2200-2900-fold sensitivity enhancement compared to those in a conventional MCZE analysis. The run-to-run repeatabilities of the migration time and peak height were good with relative standard deviations of 1.1% and 7.2%, respectively, which were better than those of normal MCZE. By applying the LVSEP technique to MCZE, a complicated voltage program for fluidic control could be simplified from four channels for two steps to two channels for one step. [ABSTRACT FROM AUTHOR]

A simple and reproducible capillazy-zone electrophoresis (CZE) method was developed for the separation and quantitalion of sulfonium-ion-containing compounds isolated from plants of the Salacia genus which are traditionally used in A urvedic medicine for the treatment of type-2 diabetes. The method sufficiently resolved four different compounds with confirmed glucosidase inhibitoiy activity, namely, salacinol, ponkoranol, kotalanol and de-O-sulfonated kotalanol. Separation could be achieved in less than 9 mm, and calibration curves showed good linearity. Detection limits were determined to be in the low pg/mL range. This method was used to demonstrate that de-Osulfonated kotalanol isolated from natural sources has identical ionic mobility to a synthetic standard. Furthermore, new extraction conditions were developed by which the zwitterionic compounds (salacinol, ponkoranol, and kotalanol) could be separated from de-O-sulfonated 1wtalanol in a single solid-phase extraction (SPE) procedure. The extraction gave reproducibly high recoveries and was used to process four commercial Salacia extracts for CZE analysis to reduce the complexity of resulting electropherograms and to facilitate the detection of the four inhibitors in question. De-O-sulfonated kotalanol was detected in two of four Salacia samples while ponkoranol was present in all four. A comparison of all samples tested demonstrated that they had remarkably similar patterns of peaks, suggesting that this CZE method may be useful in the chemical fingerprinting of Salacia-containing products. [ABSTRACT FROM AUTHOR]

Due to their high energetic profile, skeletal muscle fibers are prone to damage by endogenous reactive oxygen species (ROS), thereby causing alterations in muscle function. Unfortunately, the complexity of skeletal muscle makes it difficult to measure and understand ROS production by fibers since other components (e.g., extracellular collagen and vascular vessels) may also generate ROS. Single cell imaging techniques are promising approaches to monitor ROS production in single muscle fibers, but usually the detection schemes for ROS are not specific. Single cell analysis by capillary electrophoresis (ales chemical cytometry) has the potential to separate and detect specific ROS reporters, but the approach is only suitable for small spherical cells that fit within the capillary lumen. Here, we report a novel method for the analysis of superoxide in single fibers maintained in culture for up to 48 h. Cultured muscle fibers in individual nanoliter-volume wells were treated with tiipbenylpbosphonium hydroethidine (rPP-HE), which forms the superoxide specific reporter hydroxytriphenylphosphonium etludium (OH-TPP-E). After lysis of each fiber in their corresponding nanowell, the contents of each well were processed and analyzed by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF) making it possible to detect superoxide found in single fibers. Superoxide basal levels as well as changes due to fiber treatment with the scavenger, tiron, and the inducer, antimycin A, were easily monitored demonstrating the feasibility of the method. Future uses of the method include parallel single-fiber measurements aiming at comparing pharmacological treatments on the same set of fibers and investigating ROS production in response to muscle disease, disuse, exercise, and aging. [ABSTRACT FROM AUTHOR]

Affinity probe capilliary electrophoresis (APCE) assays, combining the separation power of CE with the specificity of interactions occurring between a target and a molecular recognition element (MRE), have become important analytical tools in many application fields. In this report, a rationalized strategy, derived from the structure-switching aptamer concept, is described for the design of a novel APCE mode dedicated to small molecule detection. Two assay configurations were reported. The first one, developed for the single-analyte determination, was based on the use of a cholesteryl-tagged aptamer (Chol-Apt) as the MRE and its fluorescein-labeled complementary strand (CS0) as the tracer (laser-induced fluorescence detection). Under micellar electrokinetic chromatography (MEKC) conditions, free CS and the hybrid formed with CholApt (duplex) were efficiently separated (and then quantified) through the specific shift of the electrophoretic mobility of the cholesteryl-tagged species in the presence of a neutral micellar phase. When the target was introduced into the preincubated sample, the hybridized form was destabilized, resulting in a decrease in the duplex* peak area and a concomitant increase in the free CS* peak area. The second format, especially designed for multianalyte sensing, employed dually cholesteryl and fluorescein-labeled complementary strands (Chol-CS*) of different lengths and unmodified aptamers (Apt). The size dependent electrophoretic separation of different Chol-CS* forms from each other and from their corresponding duplexes* was also accomplished under MEKC conditions. The simultaneous detection of multiple analytes in a single capillary was performed by monitoring accurately each target-induced duplex-to-complex change. This method could expand significantly the potential of small solute APCE analysis in terms of simplicity, adaptability, generalizability, and high-throughput analysis capability. [ABSTRACT FROM AUTHOR]

A new, commercial hybrid ceramic polymer, Ormocomp, was introduced to the fabrication of microfluidic separation chips using two independent techniques, UV lithography and UV embossing. Both fabrication methods provided Onnocomp chips with stable cathodic electro- osmotic flow which enabled examination of the Ormocomp biocompatibility by means of microchip capillaiy electrophoresis (MCE) and (intrinsic) fluorescence detection. The hydrophobic/hydrophilic properties of Onnocomp were examined by screening its interactions with bovine serum albumin and selected amino acids of varying hydrophobicity. The results show that the ceramic, organic-inorganic polymer structure natively resists bio-fouling on microchannel walls even so that the Ormocomp microchips can be used in intact protein analysis without prior surface modification. With theoretical separation plates approaching 104 m-1 for intact proteins and 106 m' for amino acids and peptides, our results suggest that Onnocomp microchips hold record-breaking performance as microfluidic separation platforms. In addition, Ormocomp was shown to be suitable for optical fluorescence detection even at near-UV range (ex 355 run) with detection limits atananomolar level (∼200 nM) for selected inherently fluorescent pharmaceuticals. [ABSTRACT FROM AUTHOR]

Gradient micro free flow electrophoresis (μFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the μFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of μFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptainer-IgE complex was estimated to be 48 ± 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HlVRT), 2:1, and 1:1 stoichiometries. [ABSTRACT FROM AUTHOR]

The rapid separation of mixtures of six peptides using porous polymer monolithic layers in electrophoresis and pressurized planar electrochromatography modes has been achieved. The separations in the former mode were performed on a generic hydrophobic poly(butyl methaciy-late-co-ethylene dimethaciylate) layer with no ionizable functionalities and required 2 mm. This layer also enabled the separation of three oligonucleotides. The separation in the pressurized planar electrochromatographic mode was carried out using a negatively charged layer prepared via cogralling of 2-aciylamido-2-methyl-1-propanesulfonic acid and 2-hydmxyethyl methacrylate on top of the generic hydrophobic monolith and was completed in 1 mm. [ABSTRACT FROM AUTHOR]

To realize efficient homogeneous electrophoretic immunoassays, we introduce discontinuous polyaciylamide gels that enable quantitative assay completion in separation lengths as short as 350 μm in <10 s. The discontinuous cross-linked gels reduce the required electrophoretic separation lengths and thereby significantly reduce the required applied electrical potentials needed to achieve 100's V/cm electric field strengths for rapid electrophoresis. To optimize the discontinuous polyacrylanilde gel assay format, we demonstrate development of a two-color homogeneous electrophoretic immunoassay for concurrent quantitation of C reactive protein (CRP) and tumor necrosis factor-α (TNF-α) for monitoring inflammatory response. To achieve necessary pore-size control at the gel discontinuity, an optimized mask-based fabrication protocol is introduced. The fabrication approach improves electrophorelic separations using the discontinuous separation gels by eliminating two confounding phenomena: (1) smaller than desired pores at the discontinuity which result in undesired physical exclusion of large-species and (2) an associated transition from small to large pores aft of the interface which acts to "destack" analyte bands during the separation. With the use of the optimized discontinuous separation gels, both assays were linear and quantitative over a two-log detection range, with a lower limit of detection of 11 ng/mL for CRP and 40 ng/ mL for TNF-α. An optimal single-point detector location was identified by balancing the separation resolution and assay duration constraints. The ultrashort separation distance electrophoretic assays developed here provide flexibility in chip and instrument design by relaxing electrical potential requirements and expanding the possibilities for assay multiplexing, therefore addressing important design considerations when developing field- portable diagnostic assays for near-patient environments. [ABSTRACT FROM AUTHOR]