Fluorescence polarization studies of affinity interactions in capillary electrophoresis.

Capillary electrophoresis (CE) combined with molecular recognition for ultrasensitive bioanalytical applications often requires the formation of stable complexes between an analyte and its binding partner. Previous studies of binding interactions using CE involve multiple-step titration experiments and are time-consuming. We describe a simple method based on laser-induced fluorescence polarization (LIFP) detection for CE separation, which allows for on-line monitoring of affinity complex formation. Because fluorescence polarization is sensitive to changes in the rotational diffusion arising from molecular association or dissociation, it is capable of providing information on the formation of affinity complexes prior to or during CE separation. Applications of the CE/LIFP method to three binding systems including vancomycin and its antibody, staphylococcal enterotoxin A and its antibody, and trp operator and trp repressor were demonstrated, representing peptide-protein, protein-protein, and DNA-protein interactions. The affinity complexes were readily distinguished from the unbound molecules on the basis of their fluorescence polarization. The relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs.     

Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis

Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies.     

Aptamer-based detection and quantitative analysis of ricin using affinity probe capillary electrophoresis

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (approximately 14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.     

Study of lipid and apolipoprotein binding interactions using vesicle affinity capillary electrophoresis

Vesicle affinity capillary electrophoresis (VCE), a newly developed technique, was designed to assess the effect of physicochemical properties of apolipoprotein (apo) on the binding to lipoproteins, under physiological conditions (phosphate-saline buffer system at pH 7.4 and 37 degrees C), using vesicle as a model. The technique results in similar lipid binding properties of apo CIII (CIII) and its peptides compared to other techniques. It also offers a fast and more sensitive tool in determining the lipid affinity of apos in a unique system simulating the dynamic binding properties of apo in vivo. A noncompetitive binding model is used to determine the multiple binding properties of CIII and its peptides to vesicle. The VCE binding constants are dependent on temperature, physicochemical properties of the protein (hydrophobicity and charge), and nature of the vesicle. The vesicles used in the VCE experiments described here have been fully characterized and found to be stable under different temperatures (4 and 37 degrees C) and voltage conditions. Migration behavior of CIII and related peptides is reported in terms of relative mobility in order to correct for variability in viscosity at different vesicle concentrations. The VCE method provides very precise data on the migration time from 0.1 to 3.3% RSD at the highest concentration of vesicle. The model and current data have been used to determine VCE binding constants and protein-to-lipid binding ratios. The model predicts that higher lipid affinity (K(B)), protein-lipid binding ratio (n), and lower protein concentration result in a shift of the binding isotherm toward a lower concentration range of vesicle. A higher vesicle mobility, reflecting the size and charge of the vesicle, results in a larger separation window between the migration time of the free protein and the complex. The value of VCE for structure-function studies and drug design for peptides and proteins that are strongly bound to lipids has been illustrated.     

Quantitative analysis of systematic errors originated from wall adsorption and sample plug lengths in affinity capillary electrophoresis using two-dimensional simulation

Two-dimensional (2D) simulation of capillary electrophoresis is developed to model affinity interaction and wall adsorption simultaneously. Finite difference schemes are used to evaluate the mass-transfer equation in cylindrical coordinates. A Langmuir second-order kinetic law is applied to regulate the wall adsorption and desorption processes. Contributions from the simulation parameters are investigated extensively, and parameters for accurate and efficient simulation are identified. With the 2D model, capillary zone electrophoresis and affinity capillary electrophoresis (ACE) in the presence of strong or weak wall adsorption are simulated to elucidate peak distortions. Finite sample injection length/amount and wall adsorption that lead to systematic errors in the estimated binding constants in ACE are quantified for the first time with both actual experiments and computer simulation. Methods for correcting the estimated binding constants are proposed to extend the usefulness of ACE.     

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis

MicroRNAs (miRNAs) are small (～22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30?000 miRNA molecules in 1 mL of serum as a potential source of nai?ve miRNAs.     

A high-efficiency cross-flow micronebulizer interface for capillary electrophoresis and inductively coupled plasma mass spectrometry.

A pneumatic nebulizer interface for capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICPMS) is reported. The interface is constructed using a high-efficiency cross-flow micronebulizer (HECFMN) and has the following features. (1) Makeup solutions can be fed to the interface by nebulizer self-aspiration and liquid gravity pressurization. (2) The liquid dead volume of the interface is approximately 65 nL, much smaller than those (200-2500 nL) reported for other interfaces. (3) The interface can be stably operated at a liquid flow rate down to 5 microL/min with a high analyte transport efficiency up to 95% to the plasma and (4) does not induce noticeable laminar flow in the CE capillary at typical nebulizer gas flow rates of 0.8-1.2 L/min. Because of these features, baseline resolution of 10 lanthanides with a CE-ICPMS system using the HECFMN interface is achieved, and detection limits and peak asymmetry are 0.05-1 microg/L and 0.93-1.23, respectively, improved significantly over those reported previously for a CE-ICPMS system using a high-efficiency nebulizer interface. Peak precision for the 10 lanthanides is in the range of 6.2-12.3% RSD (N = 5). Peak widths are from 9.1 s for 139La to 17.9 s for 175Lu. The effects of nebulizer gas flow rate, makeup solution flow rate, and spray chamber volume on CE-ICPMS signal intensity and separation are also evaluated for the HECFMN interface by the separation of Cr3+ and Cr2O7(2-).     

Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry

A microfabricated device has been developed in which electrospray ionization is performed directly from the corner of a rectangular glass microchip. The device allows highly efficient electrokinetically driven separations to be coupled directly to a mass spectrometer (MS) without the use of external pressure sources or the insertion of capillary spray tips. An electrokinetic-based hydraulic pump is integrated on the chip that directs eluting materials to the monolithically integrated spray tip. A positively charged surface coating, PolyE-323, is used to prevent surface interactions with peptides and proteins and to reverse the electroosmotic flow in the separation channel. The device has been used to perform microchip CE-MS analysis of peptides and proteins with efficiencies over 200,000 theoretical plates (1,000,000 plates/m). The sensitivity and stability of the microfabricated ESI source were found to be comparable to that of commercial pulled fused-silica capillary nanospray sources.     

Development of a multichannel microfluidic analysis system employing affinity capillary electrophoresis for immunoassay

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.     

Chiral separation of leucovorin with bovine serum albumin using affinity capillary electrophoresis.

A method for the determination of the (6R)- and (6S)-stereoisomers of leucovorin using electrokinetic chromatography (EKC) in the affinity mode has been developed. Bovine serum albumin (BSA) is used as a run buffer additive to incorporate enantiomeric selectivity into the system. Protein-wall interactions are minimized by using a poly(ethylene glycol) (PEG) coated capillary. Chiral resolution is obtained in 12.5 min with efficiencies greater than 200,000 theoretical plates using BSA as an additive, while no resolution is obtained in the absence of BSA. A general equation is derived to calculate the free energy of interaction between the leucovorin isomers and the BSA molecule. This method represents a new means of obtaining thermodynamic data for substrate binding interactions and for the general study of drug cross-reactions and interactions of drugs with serum and other proteins.     

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.     

Pressure-based approach for the analysis of protein adsorption in capillary electrophoresis

Protein adsorption to inner capillary walls creates a major obstacle in all applications of capillary electrophoresis involving protein samples. The problem is especially severe in kinetic capillary electrophoresis (KCE) techniques, which are used to study protein-ligand interactions at physiological conditions and, thus, cannot utilize extreme pH. A variety of coatings exist to reduce protein adsorption in CE, each expressing a unique surface chemistry that interacts with individual proteins differently. Here we introduce a simple pressure-based method for the qualitative assessment of protein adsorption that can facilitate the direct antiadhesive ranking of several coatings toward a protein of interest. In this approach, a short plug of the protein is injected into a capillary and propagated through with a pressure low enough to ensure adequate Taylor dispersion. The experiment is performed with a nonmodified commercial instrument in a pseudo-two-detector approach. The two detectors are mimicked by using two different distances from the capillary inlet to a single detector. If the peak area and shape do not change with changing distance, the protein does not adsorb appreciably, while a decreasing peak area with increasing distance infers inner surface adsorption. The magnitude change of the peak area between the two distances along with the overall peak shape is used to gauge the extent of protein adsorption. By using this method, we ranked antiadhesive properties of different wall chemistries for a series of proteins. The described method will be useful for optimizing protein analysis by CE and, in particular, for KCE experiments that investigate how proteins interact with their respective ligands.     

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.     

Estimation of binding constants of peptide nucleic acid and secondary-structured DNA by affinity capillary electrophoresis

An affinity capillary electrophoresis method was developed to determine a binding constant between a peptide nucleic acid (PNA) and a hairpin-structured DNA. A diblock copolymer composed of PNA and polyethylene glycol (PEG) was synthesized as a novel affinity probe. The base sequence of the probe's PNA segment was complementary to a hairpin-structured region of a 60-base single-stranded DNA (ssDNA). Upon applying a voltage, the DNA hairpin migrated slowly compared to a random sequence ssDNA in the presence of the PNA probe. This retardation was induced by strand invasion of the PNA into the DNA hairpin to form a hybridized complex, where the PEG segment received a large amount of hydrodynamic friction during electrophoresis. The binding constant between the PNA probe and the DNA hairpin was easily determined by mobility analysis. This simple method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.     

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.     

Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.     

Online concentration and affinity separation of biomolecules using multifunctional particles in capillary electrophoresis under magnetic field

To overcome several problems in affinity capillary electrophoresis (ACE), i.e., low detectability, need for sample derivatization, and difficulty in the fixation of affinity ligands (ALs), multifunctional magnetic particles (MFMPs) were prepared by immobilizing both fluorescent molecules and ALs for low-density lipoproteins onto the surface of magnetic polymer microspheres with a polyelectrolyte multilayer coating technique and applied to the ACE analysis. The prepared MFMPs showed a remarkable change in the electrophoretic mobility (mu ep) by the addition of low-density lipoproteins (LDL), whereas for high-density lipoproteins (HDL), mu ep of the MFMPs kept constant, so that it was confirmed that the MFMPs possess an affinity with LDL. On the other hand, the MFMPs can be trapped by the magnetic field even under a higher electric field for electrophoresis. By a successive on-off control of the magnetic field, online preconcentration of the LDL bound MFMPs and the selective separation of LDL from HDL were successfully achieved. In the ACE analysis of LDL employing UV detection, an 82-fold increase in the sensitivity was obtained by the on-capillary sample preconcentration using the MFMPs. When laser induced-fluorescence detection was employed, furthermore, the limit of detection for LDL was improved to the order of subpicomolar.     

Nanoinjector for capillary electrophoresis and capillary electrochromatography

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.     

Hadamard transform capillary electrophoresis

This paper reports the first demonstration of a multiplex sample injection technique in capillary electrophoresis. The sample was injected into a capillary (effective length, 4 cm) as a pseudorandam Hadamard sequence by a photodegradation technique using a high-power gating laser, and the fluorescence signal, which was measured using a probe excitation beam, was decoded by an inverse Hadamard transformation. The signal-to-noise ratio was improved by a factor of 8, which was in good agreement with the theoretically predicted value of 8.02. This approach is potentially useful for the enhancement of the sensitivity by 3 orders of magnitude in high-resolution capillary electrophoresis, combined with fluorescence detection.