Affinity assays using fluorescence anisotropy with capillary electrophoresis separation

A novel approach to detecting affinity interactions that combines fluorescence anisotropy with capillary electrophoresis (FACE) was developed. In the method, sample is injected into a capillary filled with buffer that contains a fluorescent probe that possesses low fluorescence anisotropy. If proteins or other large molecules in the sample bind the fluorescent probe, their migration through the capillary can be detected as a positive anisotropy shift. Thus, the method provides both separation and confirmation of binding to the probe. Calculations based on combining the Perrin equation and dissociation constant were used to predict the effect of conditions on aniostropy detection. These calculations predict that low probe concentrations yield the best sensitivity while higher concentrations increase the dynamic range for detection of binding partner. The assay was applied to detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe. Experimental measurements exhibited trends in anisotropy with varying probe and protein concentrations that were consistent with the calculations. The limit of detection for G(alphai1) was 3 nM when the electrophoresis buffer contained 250 nM BODIPY FL GTPgammaS. FACE affinity assay is envisioned as a method that can quantify selected binding partners and screen complex samples for compounds that possess affinity for a particular small molecule that is used as a probe.     

Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures

We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.     

Enantiomeric separation using an l-RNA aptamer as chiral additive in partial-filling capillary electrophoresis

In this paper, we report the chiral resolution of arginine using an anti-arginine l-RNA aptamer chiral selector in partial-filling CE. The effects of the capillary temperature, sample load, and aptamer plug length on the enantiomeric separation were assessed. Very high chiral resolving capability was observed at low or moderate capillary temperatures (the target peak being not detected in the separation window), whereas the practical chiral resolution was achieved only at high enough temperatures (50-60 degrees C). Over this high-temperature range, the electrophoretic behavior of the target enantiomer appeared to result from a combination of binding site heterogeneity, slow desorption kinetics, and concentration overload of aptamer binding sites. From additional thermal UV melting experiments, three RNA conformations were identified for the 50-60 degrees C temperatures. It was suggested that the presence of these different RNA conformations was a plausible source of the binding site heterogeneity.     

Effect of dextran as a run buffer additive in drug-protein binding studies using capillary electrophoresis frontal analysis

The study of drug-protein interactions by capillary electrophoresis frontal analysis requires establishment of a sufficient mobility difference between the mobility of the ligand and protein. The potential utility of dextran as a run buffer additive to manipulate the electrophoretic mobilities of low molecular weight ligands and protein in capillary electrophoresis frontal analysis binding studies was assessed. It was demonstrated that dextran was effective in improving the separation between the ligands warfarin and flurbiprofen and human serum albumin. Separation of ligand and protein increased with the concentration of added dextran (0-7.5% (w/w)), while molecular weight of the additive (70,000-2,000,000) only had a minor effect. The effect of dextran addition on viscosity and electrophoretic and electroosmotic mobilites was systematically studied. Optimal frontal analysis settings were a compromise between achieving satisfactory separation and acceptable analysis times without loss of plateau peak conditions. No effect of dextran upon the drug-human serum albumin interactions could be detected for the model ligands. Introduction of dextran into the electrophoresis buffer expands the applicability of capillary electrophoresis frontal analysis in drug research to binding interactions between proteins and low molecular weight ligands possessing similar electrophoretic mobilities.     

Determination of effective mobilities and chiral separation selectivities from partially separated enantiomer peaks in a racemic mixture using pressure-mediated capillary electrophoresis

A new measurement principle has been developed for the rapid determination of the effective mobilities of enantiomers from their partially separated peaks. The method involves (i) partial separation of the enantiomers of a racemic sample by electrophoresis, (ii) pressure mobilization of the partially separated band by the detector, (iii) calculation of the effective separation distance between the enantiomer centroids from the observed band width and the extent of theoretical band broadening, and (iv) calculation of the effective mobilities of the enantiomers (and the separation selectivity) from the effective separation distance. Experimental conditions that lead to negligible nonideal ionic contributions to the band width are outlined. The proposed method eliminates the error caused by the changing electroosmotic flow, yields complexation constant and ionic mobility values that are more precise than the conventionally obtained ones, and reduces the measurement time by 80-90%. The equations required for the calculations are presented in a simple, ready-to-use spreadsheet format.     

Western blotting using capillary electrophoresis

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.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Prediction of electrophoretic mobilities. 3. Effect of ionic strength in capillary zone electrophoresis

Plots of mobility versus the square root of ionic strength (I(1/2)) do not show the linear behavior predicted by Kohlrausch's law. Classical electrolyte theory states that such deviations are to be expected due to the finite size of the ions. This paper uses the Pitts equation to account for the effect of ionic size on the ionic strength dependence of mobilities in CZE. Experimental mobilities for carboxylates, phenols, and sulfonates of -1 to -6 charge in aqueous buffers ranging from 0.001 to 0.1 M ionic strength were described by μ(-) = μ(0) - Az (I(1/2)/(1 + 2.4I(1/2))), where the constant in the denominator is empirically determined. Infinite dilution mobilities (μ(0)) determined by extrapolation of mobility data to zero ionic strength based on this expression yielded excellent agreement (100.3 ± 3.3%) with literature values for 14 compounds in a variety of buffers. The Pitts equation provides a reasonable estimate of the constant A for solutes up to a charge of -5. However, this constant also depends on temperature and the nature of the buffer counterion, presumably due to ion association. Thus it is most appropriate to determine the constant A empirically for a given buffer system.     

Determination of shapes and maximums of analyte peaks based on solute mobilities in capillary electrophoresis

In capillary electrophoresis, the relative orders of mobilities of analyte, additive, and the complex formed determine the analyte peak shape in a way similar to the way the binding isotherms determine the peak shapes in chromatography. The three mobilities allow six possible orders; each produces a characteristic peak shape in CE. Equations describing the analyte migration in a CE system with the presence of mobility-changing additives can be implemented into computer programs to predict the migration times of the analyte peak maximums, and the predicted migration times agree well with the experimental results.     

Prediction of electrophoretic mobilities. 4. Multiply charged aromatic carboxylates in capillary zone electrophoresis

The electrophoretic mobilites of aromatic carboxylates and sulfonates at zero ionic strength were correlated with models incorporating both hydrodynamic and dielectric friction. The hydrodynamic friction was predicted using either the Hückel spherical ion model or the Perrin ellipsoidal model. Dielectric friction is the charge-induced drag caused by the reorientation of the solvent dipoles in response to the analyte charge. Based on the Hubbard-Onsager and Zwanzig expressions, the dielectric friction is related to z2/V. Expressions incorporating both the hydrodynamic and dielectric frictional terms successfully predicted infinite-dilution mobilities to within 4.4%. The influence of dielectric friction ranged from 3-8% of the overall drag for singly charged analytes to 39% of the total frictional drag for 1,2,4,5-benzenetetracarboxylate.     

Affinity capillary electrophoresis for the assessment of complex formation between viruses and monoclonal antibodies

The formation of complexes of human rhinovirus (serotype HRV2 and HRV14) with nonaggregating neutralizing monoclonal antibodies was investigated by affinity capillary electrophoresis. The method is based on preincubation of virus with antibody, followed by CE analysis. At low antibody-to-virus ratios, peaks corresponding to the complexes were broad, pointing to the presence of a heterogeneous population of virions with various numbers of antibodies bound; at a high molar ratio between virus and antibody, the peak became narrow again, indicating saturation of the 60 equivalent viral epitopes with the antibodies being attached bivalently. As SDS was used as an additive in the background electrolyte to allow for separation, its influence on complex formation was investigated. Once formed, HRV2-antibody complexes were found to be stable in the presence of the detergent but complex formation in buffer containing SDS was severely impaired. HRV14-antibody complexes were rapidly dissociated by SDS. The method proved to be useful for a rapid assessment of complex formation and might allow for an estimation of the binding stoichiometry.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated tapered fused-silica capillary with a beveled edge.

A tapered capillary tip containing a beveled edge was developed for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The optimal flow rate of a 75-microm-i.d., 90-microm-o.d. beveled tapered capillary tip was similar to a conventional flat tapered tip with a 25-microm orifice. Using a mixture of coptisine, berberine, and palmatine chloride, the sheathless CE/ ESI-MS sensitivity of a beveled 75 microm tapered tip capillary was found to be similar to a 25 microm flat tip. Although both tips offer similar CE/ESI-MS sensitivity, the beveled tapered capillary tip is more rugged and durable than a conventional 25-microm tapered capillary because of the larger outside diameter and inside diameter. To make electrical contact, the capillary tip was smeared with paint marker followed by the application of a carbon coating using a graphite pencil. Using this refined carbon-coating procedure, the capillary tip can be operated with aprotic solvents.     

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.     

Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration

A fluorescence detection system for capillary zone electrophoresis is described in which a charged-coupled device (CCD) views a 2-cm section of an axially illuminated capillary column. The CCD is operated in two readout modes: a snapshot mode that acquires a series of images in wavelength and capillary position, and a time-delayed integration mode that allows long exposure times of the moving analyte zones. By use of the latter mode, the ability to differentiate a species based on both its fluorescence emission and migration rate is demonstrated for fluorescein and sulforhodamine 101. The detection limit for fluorescein isothiocyanate (FITC) is 1.2 X 10(-20) mol; detection limits for FITC-amino acids are in the (2-8) X 10(-20) mol range.     

DNA sequencing using capillary array electrophoresis.

A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.     

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene as an additive for capillary electrophoresis

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), an aromatic-based, bowl-shaped macrocycle, was used as an additive in capillary electrophoresis. Several phenol derivatives are used as analytes to demonstrate the effect of this highly charged additive. TESMR is observed to interact differently with a mixture of positional isomers and other types of phenol derivatives. A comparative study of separations with two charged additives, TESMR and sulfobutyl ether-β-cyclodextrin (SBE-β-CD), provided insight into the selectivity exhibited by these additives. The influence of buffer pH, ionic strength, and organic modifier content on separation and peak shape is investigated. Peak asymmetry caused by the use of highly charged additives at lower pH is minimized by the addition of small amounts of a polar aprotic organic solvent to the run buffer. The effects of mixing a charged additive with a neutral additive are also discussed.     

Analysis of fission products using capillary electrophoresis with on-line radioactivity detection

Capillary electrophoresis has been used to separate metal ions characteristically associated with nuclear fission. Indirect UV absorbance and on-line radioactivity detection were used simultaneously to monitor the analytes. The radioactivity detector consists of conical plastic scintillating material with the capillary passing through the center to provide a 4π detection geometry. The wide end of the cone is optically coupled to a photomultiplier tube. Transient isotachophoretic techniques were employed to stack large volumes of samples which had low specific activities. Radioactivity detection of (152)Eu and (137)Cs was achieved at the nanocurie level for 80-100 nL injections. The detector is approximately 80% efficient, enabling samples resident in the detector window for 0.1 min to be reliably assayed. The separation of (137)Cs and (137m)Ba isotopes, which are in secular equilibrium, was modeled to demonstrate the effects of the rapid decay of (137m)Ba.     

Separation of bioconjugated quantum dots using capillary electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. Under a given set of experimental conditions, the fluorescence intensity of QDSt and QDBi emitting light at 655 nm indicated that about 90% of QDBi complexed with 70% of QDSt. A two-color experiment that made use of two different sizes of QD (i.e., 585 and 655 nm) indicated that 30% of the 655 nm QDBi complexed with 53% of the 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This, in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared.     

Determination of nucleotides in fish tissues using capillary electrophoresis

Capillary electrophoresis has been applied to quantitate nucleotide degradation in fish tissues, to provide a basis for determining the K value, an indicator of fish freshness. The three major compounds, inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx) were distinctively separated at 416 V/cm applied potential, 100 mM CAPS buffer, pH 11. There was a good correlation between the peak area and the nucleotide concentration. By using a short distance (22 cm) from the sample entrance to the detector, the identification and determination of these compounds in each sample were completed within 15 min. The results obtained correlated very well with those obtained by enzymatic assays. The capillary was completely regenerated with 1 N NaOH, to dissociate all bound materials from the capillary wall, mainly cations in the fish extract. This provided the same silica surface for repeated runs, resulting in reproducible electropherograms.