Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.     

Contactless conductivity detector array for capillary electrophoresis

A CE system featuring an array of 16 contactless conductivity detectors was constructed. The detectors were arranged along 70 cm length of a capillary with 100 cm total length and allow the monitoring of separation processes. As the detectors cannot be accommodated on a conventional commercial instrument, a purpose built set‐up employing a sequential injection manifold had to be employed for automation of the fluid handling. Conductivity measurements can be considered universal for electrophoresis and thus any changes in ionic composition can be monitored. The progress of the separation of Na⁺ and K⁺ is demonstrated. The potential of the system to the study of processes in CZE is shown in two examples. The first demonstrates the differences in the developments of peaks originating from a sample plug with a purely aqueous background to that of a plug containing the analyte ions in the buffer. The second example visualizes the opposite migration of cations and anions from a sample plug that had been placed in the middle of the capillary.     

A cam-based laser-induced fluorescence scanner for capillary array electrophoresis

Capillary array electrophoresis (CAE) is an important high throughput analytical technique. Laser-induced fluorescence (LIF) has been the dominant detection method for CAE owing to its low limit of detection (LOD) and wide linear dynamic range (LDR). Linear LIF scanners were first used in CAE because linear motions of an objective match well with a common planar array of capillaries. A problem with linear scanners is that the motor is required accelerating/decelerating so that all capillaries can be properly scanned, which makes motion control complicated and reduces the duty cycle. Rotary scanners were developed to overcome this problem. While rotary scanners have been successfully applied in CAE, the capillaries have to be arranged in a circular format, which can be inconvenient in some cases. In this report, we describe a cam-based LIF scanner as an alternative technique for CAE detection. In this system, a rotary motor is mechanically linked with a capillary holder via a cam. During operation, the motor carries the cam in a rotary motion that drives an array of capillaries on the holder to move back and forth across the objective for fluorescence detection. Using this design, the capillaries can be parallel-arranged in a plane while the motor acceleration/deceleration is avoided. To demonstrate the feasibility of this approach, we constructed a prototype instrument with a constant-velocity scanning distance of ∼10 mm, a scanning frequency of 3 Hz and a duty cycle of ∼70%. The scanner exhibited a LOD of 69 pM of fluorescein and a LDR of 3.5 orders of magnitude. Multiplexed capillary SDS-PAGE was performed on this scanner for protein separations.     

Home-made capillary array electrophoresis for high-throughput amino acid analysis

A capillary array electrophoresis (CAE) with confocal rotary scanner for high-throughput carboxytetramethylrhodamine succinimidyl ester (TAMRA)-labeled amino acid (AA) analysis is presented. Performance of the CAE setup was evaluated with AA samples. Up to 128 capillaries could be detected in parallel. For the first time, the device was applied to separate the enantiomers of isoleucine, cysteine, and glutamic acid with cyclodextrin-modified electrolytes by capillary zone electrophoresis. Baseline separation of seven AAs is also demonstrated using micellar electrokinetic chromatography method.     

Microfabricated capillary array electrophoresis DNA analysis systems

The equation of motion for a peak in a gas chromatography experiment is shown to be exact whenever the column outlet pressure is approximately zero. Based on steady-state assumptions, this equation is solved for an isothermal analysis with a single-ramp inlet-pressure or flow-rate program. Theoretical calculations of retention times are confirmed experimentally for two n-alkanes using a capillary column connected to a mass-selective detector. A slight but consistent discrepancy between theoretical and measured results is interpreted as a failure of the steady-state assumption when the magnitude of the rate of change of inlet pressure is large.     

Contaminant-induced current decline in capillary array electrophoresis

High-throughput capillary array electrophoresis (CAE) instruments for DNA sequencing suffer to varying degrees from read length degradation associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual amounts of large, slow-moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we investigate the creation and expansion of an ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. Slow-moving fragments are analytically and experimentally shown to reduce the ionic concentration of the downstream electrolyte. With injection of large fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region begins to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions, the depletion region expands, the capillary current declines dramatically, and the electrophoresis run yields a short read length or fails completely.     

DNA sequencing by capillary array electrophoresis and microfabricated array systems

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.     

Noise normalisation in capillary electrophoresis using a diode array detector

When using capillary electrophoresis with a diode array detector, the wavelength at maximum absorbance is often chosen to quantify a given analyte. However, the background noise for every wavelength should be taken into account as it is by maximising the signal to noise ratio that the lowest limit of detection will be obtained. Here, we proposed an algorithm allowing to correct an electropherogram from its background absorption and to estimate the background noise. Applying it to all the electropherograms obtained in each wavelength channel allows obtaining the background noise as a function of the wavelength, which can be used to calculate the signal to noise ratio. This not only allows selecting the best wavelength to maximise the limit of detection of a given analyte, but also to generate a noise normalised base peak electropherogram (nn-BPE). It is shown that the noise normalised base peak electropherograms substantially improve the peaks visualisation. The algorithm is part of a graphic user interface that runs under MatLab environment; it does not require any programming knowledge and is freely available.     

Integrated CMOS microchip system with capillary array electrophoresis

A complementary metal oxide semiconductor (CMOS)-capillary array electrophoresis (CAE) system has been used for DNA analysis. Because of its compactness and multiplex capability, the CAE-CMOS microchip is very suitable for the construction of a miniaturized high-throughput system for bioassays. Use of simultaneous laser-beam focusing on to the capillary array and a microscope objective contributed to the construction of the compact CMOS microchip-CAE system. To test the constructed system 100-base-pair (bp) DNA ladders and Hind III digest lambda DNA were separated in poly(vinylpyrrolidone) (PVP) sieving matrix. The miniaturized and integrated CMOS microchip system used in this work had great potential for combination with a variety of microfabricated devices for biomedical research.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.     

Combinatorial enantiomeric separation of diverse compounds using capillary array electrophoresis

Combinatorial chiral separations were performed on a 96-capillary array electrophoresis system. A comprehensive enantioseparation protocol employing neutral and sulfated cyclodextrins as chiral selectors for common basic, neutral and acidic compounds was developed. By using only four judiciously chosen separation buffers, successful enantioseparations were achieved for 49 out of 54 test compounds spanning a large variety of pK and structures. Therefore, unknown compounds can be screened in this manner to identify the optimal enantioselective conditions in just one run.     

毛细管阵列电泳技术用于L-谷氨酸消旋化反应的条件优化

L-谷氨酸消旋化反应对于制备光学纯D-谷氨酸具有重要的意义。本文利用532 nm激光诱导荧光共聚焦检测式毛细管阵列电泳仪研究了L-谷氨酸基于席夫碱中间物的消旋化反应,采用羧基四甲基罗丹明琥珀酰亚胺酯(TAMRA)荧光探针标记消旋化产物后,以含有2 mmol/L环糊精(β-CD)的100 mmol/L Tris-硼酸缓冲液(pH 10)为分离电解质进行毛细管阵列电泳分析,在该条件下TAMRA标记的谷氨酸对映体可以得到基线拆分。详细考察了醛的种类及用量、羧酸溶剂的种类、反应体系的含水量对消旋化的影响,结果表明L-谷氨酸在含20%(体积分数)水的乙酸溶剂中,以水杨醛作为催化剂(水杨醛与L-谷氨酸的物质的量比为0.2)的优化条件下可以快速地被消旋化。     

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.     

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates.

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (microCAE). In particular, the development of a novel rotary scanning confocal fluorescence detector has facilitated the high-speed collection of sequencing and genotyping data from radially formatted microCAE devices. The concomitant development of a convenient energy-transfer cassette labeling chemistry allows sensitive multicolor labeling of any DNA genotyping or sequencing analyte. High-performance hereditary haemochromatosis and short tandem repeat genotyping assays are demonstrated on these devices along with rapid mitochondrial DNA sequence polymorphism analysis. Progress in supporting technology such as robotic fluid dispensing and batched data analysis is also presented. The ultimate goal is to develop a parallel analysis platform capable of integrated sample preparation and automated electrophoretic analysis with a throughput 10-100 times that of current technology.     

Side-entry excitation and detection of square capillary array electrophoresis for DNA sequencing.

In high throughput DNA sequencing based on capillary electrophoresis, efficient coupling of the laser to each capillary is a challenge. Our group previously reported two multiple point irradiation schemes. The present work describes a more efficient excitation and detection method in which the laser light propagates through the capillary array without undergoing a serious reduction in power. An array of square capillaries (340 microns O.D. x 75 microns I.D.) was sandwiched between two fused-silica plates with an index-matching solution in between. The light was directed into the channel across the capillary array from the side. DNA sequences of PGEM/U from 24 capillaries were obtained even with a relatively low-power laser. The excitation scheme can be scaled up to hundreds of capillaries to achieve high-speed, high-throughput DNA sequencing, genetic typing and drug screening.     

Two-color excitation system for fluorescence detection in DNA sequencing by capillary array electrophoresis

Two computer-controlled galvanometer scanners are adapted for two-dimensional step scanning across a 96-capillary array for laser-induced fluorescence detection. 488 nm and 514 nm laser lines from the same Ar(+) laser were alternately coupled for two-color excitation in each capillary. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries and the excitation wavelengths. Based on the differences in absorption spectra for the dyes, the peak-height ratios in the 488 nm and 514 nm excitation electropherograms were used for peak identification for multiplexed capillary electrophoresis. Successful base calling for 24-capillary DNA sequencing was achieved to 450 bp with 99% accuracy. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components and flexibility due to the independent paths for excitation and emission.     

Design and optimization of on-chip capillary electrophoresis

We present a systematic, experimentally validated method of designing electrokinetic injections for on-chip capillary electrophoresis applications. This method can be used to predict point-wise and charge-coupled device (CCD)-imaged electropherograms using estimates of species mobilities, diffusivities and initial sample plug parameters. A simple Taylor dispersion model is used to characterize electrophoretic separations in terms of resolution and signal-to-noise ratio (SNR). Detection convolutions using Gaussian and Boxcar detector response functions are used to relate optimal conditions for resolution and signal as a function of relevant system parameters including electroosmotic mobility, sample injection length, detector length scale, and the length-to-detector. Analytical solutions show a tradeoff between signal-to-noise ratio and resolution with respect to dimensionless injection width and length to the detector. In contrast, there is no tradeoff with respect to the Peclet number as increases in Peclet number favor both SNR and separation solution (R). We validate our model with quantitative epifluorescence visualizations of electrophoretic separation experiments in a simple cross channel microchip. For the pure advection regime of dispersion, we use numerical simulations of the transient convective diffusion processes associated with electrokinetics together with an optimization algorithm to design a voltage control scheme which produces an injection plug that has minimal advective dispersion. We also validate this optimal injection scheme using fluorescence visualizations. These validations show that optimized voltage scheme produces injections with a standard deviation less than one-fifth of the width of the microchannel.