Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer drag-tag

We demonstrate the first use of a non-natural, genetically engineered protein polymer drag-tag to sequence DNA fragments by end-labeled free-solution electrophoresis (ELFSE). Fluorescently labeled DNA fragments resulting from the Sanger cycle sequencing reaction were separated by free-solution capillary electrophoresis, with much higher resolution and cleaner results than previously reported for this technique. With ELFSE, size-based separation of DNA in the absence of a sieving matrix is enabled by the end-on attachment of a polymeric "drag-tag" that modifies the charge-to-friction ratio of DNA in a size-dependent fashion. Progress in ELFSE separations has previously been limited by the lack of suitable large, monodisperse drag-tags. To address this problem, we designed, constructed, cloned, expressed, and purified a non-natural, genetically engineered 127mer protein polymer for use as an ELFSE drag-tag. The Sanger cycle sequencing reaction is performed with the drag-tag covalently attached to the sequencing primer, a major advance over previous strategies for ELFSE sequencing. The electrophoretic separation is diffusion-limited, without significant adsorption of the drag-tag to capillary walls. Although the read length (at about 180 bases) is still short, our results provide evidence that larger protein polymer drag-tags, currently under development, could extend the read length of ELFSE to more competitive levels. ELFSE offers the possibility of very rapid DNA sequencing separations without any of the difficulties associated with viscous polymeric sieving networks and hence will be amenable to implementation in microchannel and chip-based electrophoresis systems.     

Simultaneous concentration and separation of enantiomers with chiral temperature gradient focusing

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.     

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.     

Surface plasmon resonance detection for capillary electrophoresis separations

A miniaturized surface plasmon resonance sensor has been used as an on-line detector for capillary electrophoresis separations. The capillary was modified slightly to shield the sensor electronics from the high voltages applied during the separation. A three-component mixture of high refractive index materials was separated and detected at the millimolar level by an untreated gold-sensing surface. A simple protein immobilization procedure was used to functionalize the surface for selective protein detection. A hybrid buffer system was developed, in which both the deposition of immobilized protein layers and the electrophoretic delivery of protein analytes were optimized. The detection system has a reproducibility of 15%, a dynamic range of 3 orders of magnitude, and a detection limit for IgG of 2 fmol.     

Capillary gel affinity electrophoresis of DNA fragments.

The incorporation of an affinity ligand within a polyacrylamide gel provides a general means of manipulating the selectivity of capillary gel electrophoresis separations. As an example of this approach, high resolution of DNA restriction fragments by capillary gel affinity electrophoresis has been achieved by adding a soluble intercalating agent, ethidium bromide, to the gel-buffer system. A migration model has been developed that can be used for selectivity optimization. Various parameters, such as ligand concentration and applied electric field, have been examined in terms of their influence on retention and selectivity of different-size DNA molecules. From this study, high-resolution separations have been developed with efficiencies as high as 10(7) theoretical plates per meter.     

Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.     

Inline injection microdevice for attomole-scale sanger DNA sequencing

A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA sequencing separations. The microdevice comprises three axial domains for nanoliter-scale sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel. Extension fragments selectively hybridize and concentrate at the polymer interface while residual primer, nucleotides, and salts electrophorese out of the system. The plug is thermally released and injected into the CE channel by direct application of the separation voltage. To evaluate this system, 30 nL of sequencing sample prepared from 100 amol (60 million molecules) of human mitochondrial hypervariable region II amplicon was introduced into the microchip, purified, concentrated, and injected, generating a read length of 365 bases with 99% accuracy. This efficient inline injection system obviates the need for the excess sample that is required by cross-injection techniques, thereby enabling Sanger sequencing and other high-performance genetic analysis using DNA quantities approaching theoretical detection limits.     

Propagation characteristics and wavelength tuning of amplified spontaneous emission from dye-doped polymer

The propagation characteristics of amplified spontaneous emission (ASE) through a rhodamine 6 G-doped polymethyl methacrylate freestanding film waveguide were studied. This was done by shifting the excitation stripe horizontally along a transversely pumped waveguide. By this method, we could tune the ASE wavelength. The maximum tunability thus obtained was approximately 18 nm with a pump stripe length of 6 mm.     

Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis

The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four color detection channels. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in < 120 s with the microplate system. The practical utility and multicolor detection capability is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a noncovalent 2-color staining method. This work establishes the feasibility of performing high-throughput genotyping separations with capillary array electrophoresis microplates.     

Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed, and electrophoresis was a dominant transport process in the separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.     

Transmission of digital images consisting of white-light dark solitons

A distortion-free digital image is transmitted through parallel propagation of white-light photovoltaic dark solitons. The waveguide channels induced by white-light dark solitons can guide both the laser beam and the white-light beam well. We determine experimentally the critical separations of the dark solitons in the directions parallel and perpendicular to the crystalline c axis for the given crystal thickness.     

Laser-induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis

A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio approximately 250 for peaks around 500 bases. The signal-to-noise ratio is approximately 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.     

Selective detection of biogenic amines using capillary electrochromatography with an on-column derivatization technique

A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-mcirom (i.d.) fused-silica capillary tube packed with 5-microm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish.     

Capillary electrophoresis micro X-ray fluorescence: a tool for benchtop elemental analysis

A new tool was developed for separation and elemental detection by interfacing a simple capillary electrophoresis (CE) apparatus, constructed using a thin-walled fused-silica capillary, with a benchtop energy-dispersive micro X-ray fluorescence (MXRF) system. X-ray excitation and detection of the separated analytes was done using an EDAX Eagle II micro X-ray fluorescence system equipped with a polycapillary Rh target excitation source and a SiLi detector. It was demonstrated that this prototype system could be used for the separation and detection of species containing two different metals from one another, specifically Cu and Co. Free Co could also be separated from Co bound to cyanocobalamin (vitamin B-12). Two organic compounds were also separated from one another, a large biological protein, ferritin, from a small biological organic, cyanocobalamin. Preliminary average detection limits obtained on this system were on the order of 10(-)(4) M and compared favorably to those reported for the similar technique of CE-synchrotron XRF. CEMXRF allows for nondestructive, simultaneous, on-line, benchtop elemental analysis for chemical speciation applications.     

Independent optimization of capillary electrophoresis separation and native fluorescence detection conditions for indolamine and catecholamine measurements

Separation conditions in capillary electrophoresis with native fluorescence detection often represent a compromise in terms of the separation and detection figures of merit. As both the separation and fluorescence properties greatly depend on pH, the ability to independently optimize pH in the separation capillary and the detection region can improve many complex separations. When using a sheath flow cell, the pH at the detection zone can be adjusted independently of the electrophoresis buffer pH. Using capillary electrophoresis with 257-nm excitation and native fluorescence detection, more than an order of magnitude improvement in the limits of detection for dopamine (from 1400 to 120 nM) and epinephrine (from 850 to 60 nM) is achieved by maintaining the basic separation conditions and an acidified sheath buffer. The detection of dopamine in an individual Aplysia californica cerebral ganglion neuron is demonstrated.     

Interfacing capillary-based separations to mass spectrometry using desorption electrospray ionization

The powerful hybrid analysis method of capillary-based separations followed by mass spectrometric analysis gives substantial chemical identity and structural information. It is usually carried out using electrospray ionization. However, the salts and detergents used in the mobile phase for electrokinetic separations suppress ionization efficiencies and contaminate the inlet of the mass spectrometer. This report describes a new method that uses desorption electrospray ionization (DESI) to overcome these limitations. Effluent from capillary columns is deposited on a rotating Teflon disk that is covered with paper. As the surface rotates, the temporal separation of the eluting analytes (i.e., the electropherogram) is spatially encoded on the surface. Then, using DESI, surface-deposited analytes are preferentially ionized, reducing the effects of ion suppression and inlet contamination on signal. With the use of this novel approach, two capillary-based separations were performed: a mixture of the rhodamine dyes at milligram/milliliter levels in a 10 mM sodium borate solution was separated by capillary electrophoresis, and a mixture of three cardiac drugs at milligram/milliliter levels in a 12.5 mM sodium borate and 12.5 mM sodium dodecyl sulfate solution was separated by micellar electrokinetic chromatography. In both experiments, the negative effects of detergents and salts on the MS analyses were minimized.     

Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations: artificial neural network optimization for microchip analysis

Effective DNA separations in microelectrophoretic systems are complicated by the need to passivate the surface dynamically or covalently. We describe the optimization and utilization of a novel buffer system for fast DNA separations by capillary and microchip electrophoresis without the need for any surface modification or conditioning prior to separation. At concentrations as high as 5%, hydroxypropyl cellulose (HPC) has a relatively low viscosity, allowing for microchip channel filling to be performed with ease. A MES/TRIS buffer system at pH 6.1 eliminates the need for surface preconditioning procedures due to the promotion of hydrogen bonding of HPC with the wall. An additional benefit with this buffer system is the low current observed at high fields when compared to other common DNA separation buffers. An artificial neural network (ANN) was used to model the data and to predict the optimum conditions. Utility of the ANN-optimized system for molecular diagnostic testing was demonstrated by performing microchip separations on DNA samples from patients suspected of having genetic mutations associated with Duchenne muscular dystrophy (DMD). Microchip analysis easily allowed for the patient samples positive for DMD mutations to be distinguished from patient samples negative for the disease.     

边界,接头,不连续因素对一维构件扰动传播特性影响的研究

针对一维波导在边界、接头、外加激励、复杂约束情况下的行波散射规律进行了深入研究，导出了反映入射波、反射波及激励向量关系的散射矩阵表达式，对两杆波导接头、欧拉梁与杆波导接头及受到横向和转角约束的波导的反射透射系数给出了详细的推导，最后用行波法求解了封闭杆系结构的固有频率，取得了一些有价值的研究成果。     

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.     

Fritless packed columns for capillary electrochromatography: separation of uncharged compounds on hydrophobic hydrogels

A novel column is described that does not require frits to keep packing material within a capillary. A continuous bed is prepared in situ in aqueous solution by radical copolymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid (the resultant gel is denoted poly(AMPS-co-IPAAm). N,N'-Methylenebisacrylamide is used for cross-linking. On the application of an electrical field, electroosmotic flow (EOF) is developed in the bed along the capillary, where fluid propulsion would be otherwise difficult to achieve. The resultant EOF transports neutral compounds through the column without forcing the gel out of the capillary. Examination of the fluid motion in the continuous bed using a video microscope system and an image processor shows a relatively flat flow profile of EOF. The bed functions as the stationary phase for reversed-phase capillary electrochromatography (CEC). This new approach is an alternative to packed capillary columns which have been used previously in CEC. A high efficiency is obtained for a steroid which is separated on a 4.0% total monomer concentration (T), 10.0% degree of cross-linking (C), and 10.0% mole fraction of AMPS in the total monomer (S), poly(AMPS-co-IPAAm) column. A mixture of polyaromatic hydrocarbons is separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column. The capacity factor of benzo[a]pyrene increases from 0.63 to 1.91 as the acetonitrile content in a Tris-boric acid buffer is decreased from 45 to 30% (v/v). The run-to-run RSD of analyte migration time is less than 0.73%, and the day-to-day RSD is acceptable. Potential benefits of this approach are also mentioned.