Red diode laser induced fluorescence detection with a confocal microscope on a microchip for capillary electrophoresis

A highly sensitive laser induced fluorescence (LIF) detection system based on a 635 nm laser diode and cyanine-5 (Cy-5) dye, is described for use with a planar, microfluidic, capillary electrophoresis (CE) chip. The CE-chip is able to determine a protein biological threat agent simulant, ovalbumin (Ov), by performing an immunoassay separation of Cy-5 labeled anti-ovalbumin from its complex with Ov, in under 30 s. A confocal, epiluminescent detection system utilizing a photomultiplier tube gave optimum results with a 400 microm pinhole, an Omega 682DF22 emission filter, a 645DRLP02 dichroic mirror, a 634.54 +/- 5 nm excitation filter, and a Power Technology ACMO8 635 nm laser operated at 11.2 mW. Using this detector, a microchip CE device with a separation efficiency of 42,000 plates and an etch depth of 20 microm, gave a concentration detection limit of 9 pM Cy-5. This limit corresponds to the determination of 4560 injected molecules and detection of 900 of these molecules, given a probe volume of 1.6 pl and a probing efficiency of 20%.     

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.     

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.     

Mutation detection using ligase chain reaction in passivated silicon-glass microchips and microchip capillary electrophoresis

The ligase chain reaction (LCR) following PCR is one of the most sensitive and specific methods for detecting mutations, especially single nucleotide polymorphisms (SNPs). Performing LCR in microchips remains a challenge because of the inhibitory effect of the internal surfaces of silicon-glass microchips. We have tested a dynamic polymer-based surface passivation method for LCR conducted in oxide-coated silicon-glass microchips. The combination of polyvinylpyrrolidone 40 (PVP-40) at 0.75% (w/v) with an excess of the ligase produced successful LCR in the silicon-glass microchips, with yields of ligated primers comparable to reactions performed in conventional reaction tubes. Ligated primers were detected and quantified simply and conveniently using microchip capillary electrophoresis.     

Separation of a range of cations by nonaqueous capillary electrophoresis using indirect and direct detection

The use of nonaqueous media and indirect detection is reported for the separation and detection of a range of small cations. The novel applications involved separation of a range of metal ions, small nonchromophoric amines, cationic ion-pair reagents and cationic surfactants. Separations were achieved using acidified methanol containing imidazole as the UV co-ion for indirect detection. The methods produced different selectivity compared to aqueous methods using acidified aqueous imidazole solutions. Advantages of the methods include speed of analysis and prevention of sample micellerisation. The methods were shown to be quantitative and reproducible by their application to the determination of Tris content.     

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides that relies on capillary electrophoresis (CE) with laser-induced fluorescence detection is presented. This method enables complete separation of 17 GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone to improve sensitivity. The limit of detection was at the attomole level and approximately 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The relative standard deviations of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7 and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate resulting from the separate analyses of a single sample.     

Determination of glycosaminoglycan monosaccharides by capillary electrophoresis using laser-induced fluorescence detection

A newly developed capillary electrophoretic method using laser-induced fluorescence detection (CE-LIF) for the analysis of monosaccharides released from acid hydrolysis of glycosaminoglycans was studied. The method was compared with a previously published method using indirect LIF detection (CE-ILIF). For the CE-LIF method, electrophoretic conditions for the separation of the monosaccharides derivatised with 8-aminopyrene-1,3,6-trisulfonate (APTS) were optimised. The best separations were obtained using 100 mM acetate at pH 4.5 as running buffer. The influence of the injection vial volume on the precision and stability of the sample in different conditions was studied. The detection limits of the CE-LIF method were found to be 0.4-0.6 nM, while those obtained by CE-ILIF ranged from 11.4 to 14.3 microM. Other quality parameters of the method, such as run-to-run precision, day-to-day precision, and linearity were also determined. Finally, the new method was applied to the analysis of the acid hydrolysis products from a glucosaminoglycan (heparin) and a galactosaminoglycan (dermatan sulfate) and cross-contamination between the two solutions was determined. The high sensitivity of the new method allows the determination of dermatan sulfate contaminations in a heparin raw sample down to 0.04% (w/w) and broadens the practical applicability of CE-LIF for the quantitation of the endogenous levels of glycosaminoglycans in animal samples and for pharmacokinetic control after therapeutical heparin administration.     

Development of a microfabricated disposable microchip with a capillary electrophoresis and integrated three-electrode electrochemical detection

We have developed microsystems with a capillary electrophoresis and an electrochemical detector. The microfabricated CE-ECD systems are adequate for a disposable type and the characteristics are optimized for application in electrochemical detection. The system was realized by means of a polydimethylsiloxane (PDMS)-glass chip and an indium tin oxide electrode. The injection and separation channels were produced by relatively simple and inexpensive methods. A capillary electrophoresis and a three-electrode electrochemical detector were fabricated on the same substrate with the same fabrication procedure. We measured electropherograms for the testing analytes consisting of catechol and dopamine with different concentrations of 1mM and 0.1mM, respectively. The results showed an efficient and rapid separation and detection of all compounds within a very short time of around 80s using a separate electric field 60 V/cm. We could also successfully achieve an electropherogram of the separation of the 1 kb DNA ladder (8.4 ng/mul) from the 500 bp to 10 kb DNA fragments within just 150 s.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Determination of gamma-hydroxybutyric acid in biological fluids by using capillary electrophoresis with indirect detection

gamma-Hydroxybutyric acid (GHB) is a central nervous system (CNS) depressant and hypnotic which, in recent times, has shown an increasing abuse either as recreational drug (due to its euphoric effects and ability to reduce inhibitions) or as doping agent (enhancer of muscle growth). Analogues of GHB, namely gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), share its biological activity and are rapidly converted in vivo into GHB. At present, GHB and analogues are placed in the Schedules of Controlled Substances. Numerous intoxications in GHB abusers have been reported with depressive effects, seizures, coma and possibly death. The purpose of the present work was the development of a rapid analytical method based on capillary zone electrophoresis for the direct determination of GHB in human urine and serum at potentially toxic concentrations. Analytical conditions were as follows. Capillary: length 40 cm (to detector), 75 microm i.d.; buffer: 5.0 mM Na(2)HPO(4), 15 mM sodium barbital adjusted to pH 12 with 1.0 M NaOH; voltage: 25 kV at 23 degrees C; indirect UV detection at 214 nm; injection by application of 0.5 psi for 5 s. alpha-Hydroxyisobutyric acid was used as internal standard (IS). Sample pretreatment was limited to 1:8 dilution. Under these conditions, the sensitivity was approximately 3.0 microg/ml (signal-to-noise ratio >3). Calibration curves prepared in water, urine and serum were linear over concentration ranges 25-500 microg/ml with R(2)>/=0.998. Analytical precision was fairly good with R.S.D.<0.60% (including intraday and day-to-day tests). Quantitative precision in both intraday and day-to-day experiments was also very satisfactory with R.S.D.相似文献   

Assay of tramadol in urine by capillary electrophoresis using laser-induced native fluorescence detection

Capillary electrophoresis (CE) with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the direct determination of tramadol in human urine without extraction or preconcentration. The main problem in CE is the small inner diameter of the capillary which causes a low sensitivity with instruments equipped with a UV detector. Laser-induced native fluorescence with a frequency doubled argon ion laser at an excitation wavelength of 257 nm was used for the direct assay of tramadol in urine to enhance the limit of detection about 1000-fold compared to UV absorption detection. The detection system consists of an imaging spectrograph and an intensified CCD camera, which views an illuminated 1.5 mm section of the capillary. This set-up is able to record the whole emission spectra of the analytes to achieve additionally wavelength-resolved electropherograms. In the concentration range of 20 ng/ml–5 μg/ml in human urine coefficients of correlation were better than 0.998. Within-day variation determined on four different concentrations showed accuracies ranging from 90.2 to 108.4%. The relative standard deviation (RSD) was determined to be less than 10%. Day-to-day variation presented accuracies ranging from 90.9 to 103.1% with an RSD less than 8%.     

Determination of dimethylamine and other low-molecular-mass amines using capillary electrophoresis with laser-induced fluorescence detection

The potential of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the separation and determination of dimethylamine (DMA) and other low-molecular-mass amines involving precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) was investigated. Different variables that affect derivatization (pH, FITC concentration, reaction time and temperature) and separation (buffer concentration, addition of various organic modifiers, applied voltage and length of capillary) were studied. The linearity, reproducibility and reliability of the method were evaluated. The estimated instrumental detection limit for a 2-s pressure injection of the FITC-DMA derivative was 50 pg/ml (10⁻⁹ M), using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. However, for practical reasons, a minimum of 5 ng/ml DMA should be subjected to the derivatization. The applicability of the described method to the extract of atmospheric aerosol samples was demonstrated.     

Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.

The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.     

Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection

Four diuretic drugs banned in sport (amiloride, triamterene, bendroflumethiazide and bumetanide) have been separated by capillary zone electrophoresis (CZE) and detected using conventional fluorescence spectrometry. The effect of pH on electrophoretic parameters such as migration time, peak efficiency and peak height is discussed. Complete separation of the four drugs is achieved in less than 8 min at pH 8. No interference due to endogenous urine components is observed and thus direct urine analysis is feasible. Analytical figures of merit including precision and limits of detection are presented. Limits of detection range between 0.5 fmol for triamterene and 21.6 fmol for bumetanide.     

Determination of norfloxacin in rat liver perfusate using capillary electrophoresis with laser-induced fluorescence detection

A capillary zone electrophoresis method has been developed for the direct determination of norfloxacin in the physiological perfusate of isolated rat liver. Norfloxacin and the internal standard triamterene were detected using laser-induced fluorescence (LIF) detection with the excitation and emission wavelength of 325 and 435 nm, respectively. The background electrolyte (BGE) was 50 mM phosphate buffer (pH 4.6). The effect of pH and concentration of BGE on the electrophoretic migration and fluorescence response of analytes were examined. Calibration curves were linear over a wide range of 0.01-100 microg/mL. The limit of quantitation was 0.01 microg/mL. The intra- and inter-day relative standard deviation was 3.7%, or less, and the accuracy was 93.2% of the nominal concentration. No endogenous substances were found to interfere. The method was used to characterize the steady-state and transient pharmacokinetics of norfloxacin in the rat liver.     

Determination of lysophosphatidic acids by capillary electrophoresis with indirect ultraviolet detection

Lysophosphatidic acid (LPA) is the simplest form of lysophospholipid. Molecular species of LPA have been identified as the potent components in the ovarian cancer activation factor. The elevated plasma LPAs may be used as potential biomarkers for the early detection of ovarian cancer. This paper is the first report on the quantitative analysis of molecular species of LPA using capillary electrophoresis. In this work, the separation of LPAs was achieved within 14 min in an adenosine monophosphate-borate–methanol–water solution, and the measurement was accomplished by indirect UV detection. With LPA (D) as internal standard, the method had linear calibration ranges for LPAs from 2.8 to 75 μM. The detection limits for various molecular species of LPA were from 1.2 to 2.3 μM by the pressure injection at 3.45 kPa for 5 s. The method had been applied to serum fortified with LPA (S), LPA (O), LPA (P), and LPA (M) and the recoveries ranged from 83 to 112%.     

Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM