Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

Dynamic double coating,electrophoretic method with indirect detection for the simultaneous quantification of mono‐ and divalent cations in various water samples

An indirect UV detection method based on CE was developed and validated to determinate 12 metal cations, including alkali, alkaline earth, transition metal, and ammonium. In this paper, a new electrolyte system (pH 4.22) contained 20 mM benzimidazole (as co‐ion), 75 mM acetic acid (as a counter‐ion) as well as 0.6 mM 18‐crown‐6 ether was applied. The metal ions were completely separated within 8 min under hydrodynamic mode injection with a running voltage of 20 kV at 25 ± 0.1°C. Additional use of the dynamic double coating method enabled to get an excellent repeatability of migration times and quantitative parameters for all analytes. The repeatability of migration times for analytes were less than 0.9% and peak areas and peak heights ranged from 3.7 to 7.2 and 3.9 to 7.7%, respectively (n = 6). The proposed technique proved to be definitely faster and less expensive in comparison to currently employed methods. In this work, we discuss also the linear range, method detection limits as well as precision and accuracy. The applicability of the elaborated method was authenticated by the quantification of metal ions in commercially available mineral water, tap water, and selected medical injection samples.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

High‐speed separation and detection of amino acids in laver using a short capillary electrophoresis system

A high‐speed separation method of capillary MEKC with LIF detection had been developed for separation and determination of amino acids in laver. The CE system comprised a manual slotted‐vial array (SVA) for sample introduction that could improve the separation efficiency by reducing injection volume. Using a capillary with 80 mm effective separation length, the separation conditions for amino acids were optimized. Applied with the separation electric field strength of 300 V/cm, the ten amino acids could be completely separated within 2.5 min with 10 mol/L Na₂HPO₄–NaOH buffer (pH = 11.5) including 30 mmol/L SDS. Theoretical plates for amino acids ranged from 72 000 to 40 000 (corresponding to 1.1–2.0 μm plate heights) and the detection limits were between 25 and 80 nmol/L. Finally, this method was applied to analyze the composition of amino acids in laver and eight known amino acids could be found in the sample. The contents of five amino acids, tyrosine, glutamic acid, glycine, lysine, and aspartic acid that could be completely separated in real sample were determined. The recoveries ranged from 82.3% to 123% that indicated the good reliability for this method in laver sample analysis.     

Separation and determination of flavonoids in Agrimonia pilosa Ledeb. by capillary electrophoresis with electrochemical detection

Five flavonoids (catechin, hyperoside, quercitrin, quercetin, and rutin) were separated and determined by capillary electrophoresis with electrochemical detection. Effects of several important factors, such as the pH and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to determine the optimum conditions. The five flavonoids were baseline separated within 20 min in a 60 cm length capillary at a separation voltage of 19.5 kV with a running buffer consisting of 60 mmoL/L Na2B4O7 - 120 mmoL/L NaH2PO4 (pH = 8.8). The relationship between peak current and analyte concentration was linear over about two orders of magnitude with detection limits (S/N = 3) ranging from 0.02 to 0.05 microg/mL for all compounds. This method was successfully used to determine the above five flavonoids in Agrimonia pilosa Ledeb. with relatively simple extraction procedures, and the assay results were satisfactory.     

Capillary electrophoresis of DNA in agarose solutions at 40 degrees C.

DNA fragments ranging from 72 to 1353 bp in length (phi X174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3-2.0% solutions of agarose (Sea-Plaque GTG) at 40 degrees C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16 s. The internal capillary diameter was 150 mu, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb-sized circular DNA separations in agarose solutions, and those of Mb-sized DNA-agarose complexes in linear polyacrylamide solutions.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

Simple in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin

An in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2‐morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow‐injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r²) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.     

毛细管电泳-安培检测法用于7-甲基鸟苷与丝裂霉素C分离检测的研究

建立了一种同时分离检测7-甲基鸟苷与丝裂霉素C的毛细管电泳-安培检测方法。在950 mV电极(工作电极:0.3 mm微型石墨圆盘电极;参比电极:Ag/AgCl;辅助电极:Pt丝)电位下,于20 mmol/L的磷酸盐缓冲体系(pH 9.4)中,采用18 kV的分离电压进行分离。在最佳条件下,7-甲基鸟苷与丝裂霉素C在10 min内实现分离,7-甲基鸟苷与丝裂霉素C的线性范围均为0.50~50 mg/L,检测限分别为0.050 mg/L与0.025 mg/L。将该方法用于模拟尿样和模拟兔血清样的检测,7-甲基鸟苷与丝裂霉素C的回收率为93.0%～97.2%,结果令人满意。     

Further Study on Separation of DNA Fragments by Capillary Electrophoresis by Quasi-interpenetrating Network of Polyacryamide and Polyvinylpyrrolidone with UV Detection

Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.     

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra‐small detection cell length

Narrow peaks are important to high‐resolution and high‐speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra‐small detection cell length of 0.36 μm was easily achieved by using an image sensor.     

Short-end injection capillary electrophoresis for quantification of plasma chondroitin sulfate isomer disaccharides

We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Δdi-0S and Δdi-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.     

Improvement of reproducibility and sensitivity of CE analysis by using the capillary coated dynamically with carboxymethyl chitosan

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis.     

Determination of rhein and physcion in rhubarb by microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection

Microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection was developed for the separation and detection of physcion and rhein in rhubarb. In contrast to the conventional capillary electrophoresis method, ammonium acetate-dimethyl sulfoxide was used as the basic buffer system in this method. The effects of background buffer, buffer apparent pH*, buffer concentration, water ratio, sample preparation method, and separation voltage on separation and detection were investigated. Optimized separation and detection conditions were obtained: the buffer consisted of 20 mmol/L of ammonium acetate in hydro-organic solvent composed dimethyl sulfoxide, formamide, and water mixed at 60/20/20 (v/v/v) ratio. The separation voltage was 1.9 kV. Under these conditions, the physcion, rhein, and other components of rhubarb can be completely separated within 150 s. Under the methodological verification, good linearity (R ≥ 0.9995) for physcion and rhein, and low limits of detection (0.085 μg·mL⁻¹ and 0.077 μg·mL⁻¹, respectively), satisfactory peak area precisions, migration time precisions (1.74%–3.09%), and accuracy (recovery rate 97.8% and 101.4%) were achieved. It is shown that the proposed method is simple, efficient, fast, sensitive, simple instrument, consumes few samples, has low operating cost, and is linear.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

Quaternary ammonium chitosan derivative dynamic coating for the separation of veterinary sulfonamide residues by CE with field-amplified sample injection

A quaternary ammonium chitosan, 2-hydroxypropyltrimethylammonium chloride chitosan (HACC), has been developed for the dynamic coating material in CE for the first time. It presented many advantages such as favorable water solubility, satisfactory coating efficiency, and EOF toward the anode at pH >7.0. Using the modified fused-silica capillary, sulfonamides (SAs), an important group of veterinary drugs, were separated and detected by CE combined with field-amplified sample injection (FASI). The LODs of sulfonamides with UV detection were less than 0.5 ng/mL. The proposed method has been applied to the determination of veterinary sulfonamide residues in samples such as chicken, beef, and honey with fast separation (15 sulfonamides within 20 min), low LODs (0.1-0.5 ng/mL), and good reliability compared to the criteria of China (GB/T 18932.17-2003).     

Dispersive micro‐solid‐phase extraction combined with online preconcentration by capillary electrophoresis for the determination of glycopyrrolate stereoisomers in rat plasma

A simple and sensitive analytical method for four isomers of glycopyrrolate in rat plasma was developed using cation‐selective exhaustive injection‐sweeping cyclodextrin‐modified electrokinetic chromatography (CSEI‐Sweeping‐CDEKC) for online enrichment combined with dispersive micro‐solid‐phase extraction pretreatment. The CSEI‐Sweeping‐CDEKC was conducted on an uncoated fused silica capillary (40.2 cm × 75 μm) with an applied voltage of –20 kV. The electrophoretic analysis was carried out in 30 mM phosphate solution at pH 2.0 containing 20 mg/mL sulfated‐β‐cyclodextrin and 5% acetonitrile. Under these optimized conditions, the detection limit for racemic glycopyrrolate was found to be 2.0 ng/mL and this method could increase 495‐fold detection sensitivity compared with the traditional injection method. Additionally, the parameters that affected the extraction efficiency of dispersive micro‐solid‐phase extraction were also examined systematically. The glycopyrrolate isomers in rat plasma samples as low as 0.0625 μg/mL were able to be separated and detected by capillary electrophoresis with the aid of CSEI‐sweeping. The findings of this study show that the dispersive micro‐solid‐phase extraction pretreatment coupled with CSEI‐Sweeping‐CDEKC is a rapid and convenient method for analyzing glycopyrrolate isomers in rat plasma.     

Electrokinetic injection of samples into a short electrophoretic capillary controlled by piezoelectric micropumps

Electrokinetic sample injection using two piezoelectric micropumps has been proposed for electrophoresis in short capillaries. The sample is brought to the injection end of the capillary using one of them. Then, the high‐voltage source is turned on and the sample is injected electrokinetically for a defined time. The injection is terminated by removal of the sample zone by the flowing separation electrolyte pumped by the second piezoelectric micropump. The RSD value, expressing the repeatability of the injection, does not exceed 4%. The injection apparatus does not contain any mobile mechanical components, there is no movement of the capillary and both its ends remain constantly in the solution during both the sample injection and separation. Thus, the micropumps replace the six‐way injection valve and linear pump in similar types of injection apparatuses. The injection was tested in the separation and determination of ammonium and potassium ions in two samples of mineral fertilizers. The separation was performed in background electrolyte containing 500 mM of acetic acid + 20 mM Tris + 2 mM 18‐crown‐6 (pH 3.3) in a capillary with id 50 μm and total length/length to the contactless conductivity detector of 10.5/8 cm. The injection and separation took place at a voltage of 5 kV and the separation time equaled 20 s. The measured values of the analyte contents corresponded to the value declared by the manufacturer within the reliability interval, where RSD equaled between 3.5 and 4.7%.     

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

Determination of sulfa antibiotic residues in river and particulate matter by field-amplified sample injection-capillary zone electrophoresis

In the present research, field-amplified sample injection–CZE (FASI–CZE) coupled with a diode array detector was established to determine trace level sulfa antibiotic. Sulfathiazole, sulfadiazine, sulfamethazine, sulfadimethoxine, sulfamethoxazole, and sulfisoxazole were selected as analytes for the experiments. The background electrolyte solution consisted of 70.0 mmol/L borax and 60.0 mmol/L boric acid (including 10% methanol, pH 9.1). The plug was 2.5 mmol/L borax, which was injected into the capillary at a pressure of 0.5 psi for 5 s. Then the sample was injected into the capillary at an injection voltage of –10 kV for 20 s. The electrophoretic separation was carried out under a voltage of +19 kV. The capillary temperature was maintained at 20˚C throughout the analysis, and six sulfonamides were completely separated within 35 min. Compared with pressure injection-CZE, the sensitivity of FASI-CZE was increased by 6.25–10.0 times, and the LODs were reduced from 0.2–0.5 to 0.02–0.05 μg/mL. The method was applied to the determination of sulfonamides in river water and particulate matter samples. The recoveries were 78.59–106.59%. The intraday and interday precisions were 2.89–7.35% and 2.77–7.09%, respectively. This provides a simpler and faster method for the analysis of sulfa antibiotic residues in environmental samples.