Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.     

Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: a critical comparison

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.     

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.     

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution(SINGLEBOND)capillary electrophoresis (PS(SINGLEBOND)CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0(SINGLEBOND)23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. (c) 1996 John Wiley & Sons, Inc.     

Electrophoresis of long DNA molecules in linear polyacrylamide solutions

Electrophoresis of long DNA (T4 DNA; 166 kb, S. pombe chromosomal DNA; 3-6 Mb) in linear polyacrylamide solutions was investigated by fluorescence microscopy and capillary electrophoresis. In the past studies on electrophoresis of long DNA in a polymer solution, it was reported that DNA migrates in 'U-shape conformation'. We found that at higher polymer concentrations, the shape of the migrating DNA changes from U shape to linear shape ('I-shape conformation'). In the migration mode with the I-shape conformation, the DNA moves with almost constant velocity and constant shape. However, the migration velocity does depend on the DNA size, and it is possible to separate DNAs under this I-shape motion. Actually, Mb-sized DNAs are well separated within 5 min in the region for the I-shape motion by means of capillary electrophoresis with a DC field. Considering that it takes 20 h to separate Mb-sized DNAs by standard pulsed-field gel electrophoresis (PFGE), this results will be useful for the separation of giant DNAs.     

Electrochemical profiles of Herba Saussureae Involucratae by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection method has been developed for the determination of the pharmacologically active ingredients, acacetin, rutin, umbelliferone, kaempferol, apigenin, luteolin and quercetin, in Herba Saussureae Involucratae. Under optimum conditions, the seven analytes could be completely separated within 19 min in a 75 cm length capillary at a separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode, positioned opposite the outlet of the capillary in a wall-jet configuration at a potential of +950 mV (vs a saturated calomel electrode) was used as the working electrode. A good linear relationship was established between peak current and concentration of the analytes over two orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 1.2 x 10(-7) to 4.1 x 10(-8) g/mL for all analytes. The proposed method has been successfully applied to the analyses of bio-active components of Herba Saussureae Involucratae samples after a relatively simple extraction procedure. The assay results show that the resultant electrochemical profiles are indicative of the content diversity of each electrochemically active ingredient in the various samples, and may also offer some evidence for phytotaxonomy.     

Determination of josamycin in rat plasma by capillary electrophoresis coupled with post-column electrochemiluminescence detection

A novel determination method for josamycin (JOS) based on capillary electrophoresis-electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 microm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 microm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3(2+) and 50mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 microg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.     

Separation procedures capable of revealing DNA adducts

Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32P-postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.     

Detection of DNA fragmentation and endonucleases in apoptosis

DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Fast molecular diagnostics of canine T-cell lymphoma by PCR and capillary gel electrophoresis with laser-induced fluorescence detector

Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor gamma (TCRgamma) gene. After the PCR amplification of the specific TCR( gene in dogs, the 90-bp DNA fragment amplified was separated in a fused-silica capillary by CGE-LIF. Under an electric field of 375 V/cm and with a sieving matrix of 1.5% poly (ethyleneoxide) (M(r) 600,000), the amplified PCR products were analyzed within 4 min by CGE separation. When the CGE-LIF method was applied to real clinical samples of the specific DNA fragment of the TCR( gene, the migration time and the corrected peak area showed relative standard deviations (n=5) of 0.29% and 0.58%, respectively. Both methods of CGE-LIF and slab gel electrophoresis showed same results for nine clinical samples. This PCR/CGE-LIF technique may prove to be a new fast and simple tool for the rapid diagnosis of the PCR-amplified DNA of canine T-cell lymphoma.     

Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection

The capillary electrophoresis (CE) system with optical fiber light-emitting diode (optical fiber LED) induced fluorescence detector was developed for the analysis of the excitatory amino acids (EAAs) tagged with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm x 75 microm i.d.) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV. High sensitivity was obtained by the use of optical fiber LED induced fluorescence detector with a violet LED as the excitation light source. The limits of detection (S/N = 3) for glutamic acid (Glu) and aspartic acid (Asp) were 2.1 x 10(-8) and 2.3 x 10(-8) M, respectively. The detection approach was successfully applied to the analysis of Glu and Asp in biological fluids including human serum, rabbit serum and human cerebrospinal fluid (CSF) with satisfactory results.     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) using a noncompetitive format has been developed. In this method, antigen (Ag) reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ag-Ab* complex produced in the solution are separated by capillary zone electrophoresis in a separation capillary. Then they catalyze enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB(Red)) and H(2)O(2) in a reaction capillary following the separation capillary. The reaction product, TMB(Ox), can be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, a significant amount of TMB(Ox) can be produced for detection. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. A tumor marker (CA15-3) was used as a model, in order to test the method. The concentration LOD of CA15-3 is 0.024 U/ml, which corresponds to a mass detection limit of 1.3x10(-7) U.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

A micromachined capillary electrophoresis chip with fully integrated electrodes for separation and electrochemical detection

A novel analytical microsystem with fully integrated electrodes for electrophoresis and amperometrical detection is described. With respect to the lab-on-a-chip concept a capillary electrophoresis (CE) microsystem has been fabricated with a total of six gold electrodes for sample injection, separation and electrochemical detection using standard microfabrication technologies. The device is a ready-to-use system that does not need any extra mechanical apparatus for electrode insertion. The CE-chip has successfully been tested by measuring hydrogen peroxide, ascorbic acid and uric acid simultaneously. All three oxidizable species could be detected in less than 70 s. Glucose was detected by performing an enzymatic reaction along the separation channel. The microsystem showed a very good reproducibility.     

Indole-3-acetic Acid Levels of Plant Tissue as Determined by a New High Performance Liquid Chromatographic Method

A method for the analysis of indole-3-acetic acid (IAA) in plant extracts has been developed based on high performance liquid chromatography separation of IAA on a microparticulate strong anion exchange column followed by quantitation with two selective detectors: an electrochemical, carbon paste amperometric detector and/or a fluorescence detector. The detection limit for IAA is less than 1 nanogram with the fluorescence detector and less than 50 picograms with the electrochemical detector.

The IAA levels are reported for various tissues of wheat, pinto beans, soybeans, cotton, and corn.

     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.