Capillary Electrophoresis Running Buffer Research Articles

Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes.

Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.

High Affinity γPNA Sandwich Hybridization Assay for Rapid Detection of Short Nucleic Acid Targets with Single Mismatch Discrimination

Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

Simultaneous Separation of Inorganic Ions and Neutral Organics on a Sol-Gel-Coated Capillary using Combined Capillary Zone Electrophoresis and Capillary Electrochromatography

Capillary zone electrophoresis was combined with capillary electrochromatography to effect the separation of ionic and neutral organic mixtures. Sol-gel technology was used to coat the capillary wall with a silane-based stationary phase. An n-octyl phase was used for cation-organic mixtures and an octadecyl phase, which incorporated a quaternary ammonium group to effect electroosmotic flow reversal, was employed for anion-organic mixtures. Tetraethoxy silane was used as a spacer to control the density of stationary phase interactive groups. Some data on the optimum ratio of analyte-interactive group to spacer silane in the sol-gel reaction mixture was obtained. Mobile phases were mixtures of typical capillary electrophoresis run buffer and reversed phase chromatography constituents. In all cases, the analytes were separated both within and between classes, the ions exiting the capillary as a group before the neutral organics. Investigation of mobile phase methanol cosolvent and pH effects found that electrophoretic and chromatographic behavior in the mixed mode system was what would be observed in a single mode separation, demonstrating the independence of the two modes. Separation of ions and neutrals in a red wine and multivitamin is demonstrated.

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K 3[Fe(CN) 6]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10 −4 M luminol added to the CE running buffer and 1.0 × 10 −4 M K 3[Fe(CN) 6] in 0.2 M NaOH solution introduced postcolumn. The proposed CE–CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10 −7 M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10 −7 to 3.0 × 10 −5 M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.

Separation of Bioconjugated Quantum Dots Using Capillary Electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. Under a given set of experimental conditions, the fluorescence intensity of QDSt and QDBi emitting light at 655 nm indicated that about 90% of QDBi complexed with 70% of QDSt. A two-color experiment that made use of two different sizes of QD (i.e., 585 and 655 nm) indicated that 30% of the 655 nm QDBi complexed with 53% of the 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This, in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared.

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K 3[Fe(CN) 6]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10 −5 M luminol added to the CE running buffer and 5.0 × 10 −5 M K 3[Fe(CN) 6] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10 −8 to 2.5 × 10 −6 M ( r = 9991), and a detection limit of 2.0 × 10 −8 M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10 −7 M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.

Site-specific sampling of taurine from rat brain followed by on-line sample pre-concentration, throughout in-capillary derivatization and capillary electrophoresis

A method of pinpoint-sampling followed by on-line pre-concentration of the sample, throughout in-capillary derivatization and capillary electrophoretic separation was evaluated by demonstrating the detection of taurine, 2-aminoethanesulfonic acid at a specific location of a rat brain. The direct sampling of taurine from the rat brain was accomplished by using voltage injection associated with two kinds of driving forces, electrophoretic flow and electroosmotic flow (EOF). The capillary tube (75 microm of inner diameter x 375 microm of outer diameter) of the capillary electrophoresis (CE) apparatus was already filled with a CE run buffer, viz., 40 mM phosphate-borate buffer (pH 10) containing 2mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent. One end of a platinum wire (0.5mm o.d.), used as the anode, and the inlet end of capillary tube (from which a 1.0 cm long polyimide coating was removed), were pricked down onto the surface of either the cerebrum or cerebellum of a rat brain at a location of very small dimension. When a low voltage (5 kV, 30s) was applied, taurine began to move from the rat brain into the capillary tube, and, simultaneously, electric focusing of taurine occurred by the action of "the pH-junction effect" at the inlet end of the capillary tube. After completing the injection, both the platinum wire and capillary tube were detached from the brain and dipped into the run buffer in an anode reservoir filed with the same solution as that in the capillary tube for the CE apparatus. Then, by applying a high voltage (20 kV) between the ends of the capillary tube, taurine was automatically derivatized to yield the fluorescent derivative, separated and detected with fluorescence (E(x)=340 nm, E(m)=455 nm) during migration throughout the capillary tube. The migration profiles obtained from cerebrum and cerebellum appeared to be different, but the peak corresponding to taurine was identified on both electropherograms. The efficacy of the present method including sample on-line pre-concentration prior to throughout in-capillary derivatization CE was first verified with several preliminary experiments by using samples of taurine in water, saline and a piece of 1.5% agar-gel block, as an alternate standard for the rat brain used in this study.

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.

Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.

Using DNA-binding proteins as an analytical tool.

We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe-target complex (P.T) and unbound P are determined. Distinguishing between P and P.T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P.T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P.T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P.T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P.T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.

Enantioseparation of underivatized amino acids by capillary electrophoresis using copper(II)-( S)-3-aminopyrrolidine- l-histidine ternary complex as the chiral selector

The enantiomeric ternary complex formed by Cu(II), ( S)-3-aminopyrrolydine, and l-histidine is shown to be an effective chiral selector for separation of underivatized amino acid enantiomers by capillary electrophoresis (CE). None of the constituent binary complexes, i.e. Cu(II)-( S)-3-aminopyrrolidine and Cu(II)- l-histidine, exhibit the same stereoselectivity. Further, the chiral separation is significantly enhanced in the presence of polyvinyl alcohol (PVA), a linear polymer, in the CE running buffer.

Analysis of Common Household Cleaner-Disinfectants by Capillary Electrophoresis

The use of capillary electrophoresis (CE) as an analytical technique in research, industrial, and commercial laboratories is growing rapidly. It is therefore very important to expose undergraduate instrumental analysis students to capillary electrophoresis. In this report we describe the CE analysis for benzalkonium compounds in common household cleaners and disinfectants. The surfactant nature of the benzalkonium compounds is the key consideration in performing the analysis, and modifications to the CE running buffer must be performed in order to successfully analyze the products. This experiment also illustrates the importance of (i) using peak areas corrected for variations in migration time to improve accuracy and (ii) using internal standards to improve the precision of capillary electrophoresis results.

CAPILLARY ELECTROPHORESIS DETERMINATION OF BUTALBITAL FROM SERUM USING SOLID PHASE EXTRACTION (SPE) AND ULTRAVIOLET DETECTION

Butalbital is a sedative hypnotic that is used mainly in combination with analgesics in the treatment of tension headaches. This paper develops and validates a rapid and sensitive method for the determination of butalbital in serum using capillary electrophoresis (CE) and ultraviolet detection. The internal standard chosen for this assay was aprobarbital because it showed similar chemical structure, identical chromatographic/SPE characteristics, and was commercially available. The CE run buffer contained 50 mM sodium dihydrogen phosphate buffer at pH 9.0 containing 20 mM hydroxypropyl-β-cyclodextrin as a capillary zone electrophoresis (CZE) buffer additive. An SPE method, using C[18] Bond-Elut cartridges, was developed to recover the drug and internal standard from serum. The CE system consists of a 75 micron I.D., 52 cm length fused silica capillary maintained at a run voltage of 15 kV with sample detection performed at 254 nm. The limit of detection for butalbital was <l μg/mL from serum. Linear calibration curves generated in the concentration ranges of 1 to 60 μg/mL showed a coefficient of determination greater than 0.99. The precision and accuracy of the method calculated as RSD and error were 0.65–3.16% and 2.20–8.90%, respectively. Butalbital is commonly coadminisetred with aspirin, acetaminophen, caffeine, and codeine for the treatment of tension headaches. The CE method baseline resolved these compounds from the butalbital and the internal standard. Therefore, there is a decreased chance for interference from commonly coadministered compounds in this assay.

Separation of amino acid and peptide stereoisomers by nonionic micelle-mediated capillary electrophoresis after chiral derivatization

Enantiomers of amino acids and peptides were derivatized with a fluorescent chiral reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [ R-(−)- or S-(+)-NBD-PyNCS] and the resulting diastereomeric derivatives separated by capillary electrophoresis (CE). The CE running buffer consisted of 25 m M acetate buffer (pH 4) and 10 m M of the nonionic surfactant Triton X-100. The excitation maximum of NBD-PyNCS at 480 nm matches the major Ar-ion emission line at 488 nm allowing sensitive laser-induced fluorescence detection with limits of detection around 50 n M. d-Proline and d-aspartate spiked (at 10 −4 M and 10 −5 M concentrations, respectively) into complex biological matrices (rabbit serum and homogenate of Aplysia californica buccal ganglion) are detected without matrix interferences. This method has also been applied to the determination of d- and l-amino acid residues in peptides after acid hydrolysis. Results from the chiral analysis of the naturally-occurring peptide, gramicidin D, are shown.

Membrane-Based On-Column Mixer for Capillary Electrophoresis/Electrochemistry

One of the drawbacks of chemically modified electrodes for electrochemical detection in conjunction with capillary electrophoresis (CE) is that the optimal run buffer for the CE separation is often not compatible with the electrolyte conditions necessary for maximum detector response. This paper reports the development of a membrane-based on-column mixer for CE which makes it possible to perform the separation and detection using different buffer conditions. The mixer consists of a cellulose acetate tube ca. 2-mm length and 50-mm i.d. The pH and/or ionic strength of the CE run buffer can be altered at the detector end by placing the mixer in a cathodic buffer reservoir containing a modifying solution of either HCl or NaOH