Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Analysis of DNA complexes with small solutes by CE with LIF detection

In this article, we describe the analysis of aptamers for Hg²⁺ ions through CE with LIF (CE‐LIF) detection using 2% poly(ethylene oxide) solutions containing OliGreen (fluorophore). In the presence of an EOF, DNA strands migrating against the EOF were detected at the cathode end. Four DNA strands – T₃₃, T₅C₂₈, T₅C₅T₂₃, and T₁₅C₅T₁₃ – could not be separated through CE‐LIF in the absence of Hg²⁺. At 0.3 mM Hg²⁺, however, all four were partially separated within 20 min, with SDs of the migration times all being less than 2.5%. From the CE, fluorescence, and ellipticity data, we concluded that the conformations of these four DNA strands all changed from random‐coil to folded structures as a result of T–Hg²⁺–T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T₇, T₁₅, and T₃₃ in the absence and presence of Hg²⁺, indicating various interactions of the DNA strands with Hg²⁺. Using this simple, high‐resolution CE approach, we also demonstrated that adenosine triphosphate has a stronger interaction with the adenosine triphosphate aptamer than with either the platelet‐derived growth factor aptamer or T₃₃. This CE approach holds great potential for screening aptamers for small solutes, studying the catalytic activity of DNAzymes, and evaluating the biological functions of microRNA.     

RNA analysis by MEKC with LIF detection

We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

Improved peak capacity for CE separations of enzyme inhibitors with activity‐based detection using magnetic bead microreactors

A technique for separating and detecting enzyme inhibitors was developed using CE with an enzyme microreactor. The on‐column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase‐coated superparamagnetic beads (2.8 μm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme microreactor was detected by LIF. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation. The goal of this study was to improve peak capacities for inhibitor separations relative to previous studies, which combined continuous engagement electrophoretically mediated microanalysis and transient engagement electrophoretically mediated microanalysis to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Peak capacities were increased to ≥20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. Five reversible inhibitors of alkaline phosphatase (theophylline, vanadate, arsenate, L ‐tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures.     

Dynamic modification of poly(methyl methacrylate) chips using poly(vinyl alcohol) for glycosaminoglycan disaccharide isomer separation

We describe a microchip electrophoresis (MCE) method for the assay of unsaturated disaccharides of chondroitin sulfates, dermatan sulfates, and hyaluronic acid (HA). Poly(vinyl alcohol) (PVA) could be irreversibly adsorbed onto poly(methyl methacrylate) (PMMA) substrates and this approach was applicable for dynamic coating. The characteristics of the PMMA surface with PVA coating were evaluated in terms of the wettability, EOF, and adsorption of 2-aminoacridone (AMAC)-labeled disaccharide. The water contact angle decreased from 73 degrees on a pristine PMMA surface to 37.5 degrees on a PVA-coated surface, indicating that the PVA coating increased hydrophilicity. EOF was reduced approximately twofold and was relatively stable. Scanning electron microscopy and fluorescence microscopy images showed that adsorption of AMAC-labeled disaccharides was dramatically suppressed. Using the PVA coating, baseline separation of two pairs of glycosaminoglycan (GAG) disaccharide isomers, DeltaDi-diS(B)/DeltaDi-diS(D) and DeltaDi-0S/DeltaDi-HA, was achieved in Tris-borate buffer within 130 s by MCE.     

A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

A simple method for producing a sheath flow cuvette in PDMS suitable for post‐column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross‐section of approximately 635 μm diameter, whereas the second cuvette had a 300×300 μm² square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300×300 μm² square sheath flow channel to expand uniformly and accommodate the larger 330–340 μm od round separation capillary, producing a self‐aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15–30 zmol fluorescein detection limits were obtained for 12 and 50 μm id capillaries.     

Simultaneous LIF and retro-reflected beam interference detection for CE

This Short Communication describes a novel optical detection method for CE, based on the combination of LIF detection and retro-reflected beam interference detection. By the use of a side-illuminated laser beam, an on-column multifunctional detection for CE has been developed, and some key elements in the scheme have been optimized. In addition, two miscellaneous samples including fluorescent dyes, carbohydrates and amino acids have been determined to evaluate its performance. Without any additional pretreatment, sufficient LOD and linear ranges have been achieved for most analytes. Its performance in retro-reflected beam interference detection is better than those mentioned in a former report, and its fluorescent sensitivity is comparable with the achievable sensitivity by conventional LIF systems, all of which together combine high sensitivity and universal analysis to a certain extent.     

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17?min, while the shortest one separates 9 isoforms in < 8?min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.     

CE coupled with amperometric detection using a boron-doped diamond microelectrode: validation of a method for endogenous norepinephrine analysis in tissue

The level of endogenous norepinephrine (NE) in several tissue types was determined by CE with amperometric detection. We report herein on the method validation by HPLC using both amperometric and coulometric detection (CD). Keys to the method were the use of a diamond microelectrode for detection and off-line SPE for sample preparation. The run buffer was a 250 mM borate solution adjusted to pH 8.8 with potassium hydroxide. The diamond microelectrode exhibited a low and stable background current, and a low peak-to-peak noise < or =0.65 pA at the detection potential of +0.86 V versus Ag/AgCl. For standard solutions, the detector signal (i.e., oxidation current) changed linearly with the NE concentration (r(2) = 0.999) between 60 and 1000 nmol/L with an estimated LOD of 51 nmol/L (S/N = 3) and a response variability of 4.5% (RSD, n = 5). An Oasis MCX sorbent was used for SPE and the procedure produced an NE recovery of 95.1 +/- 5.6% (n = 6) from tissue homogenates. NE levels in the spleen, small intestine, and heart of a normotensive rat were found to be in the range of 0.77-0.97, 0.22-0.32, and 0.29-0.45 microg/g tissue (n = 3), respectively.     

On-column detection of multiphoton-excited fluorescence in CE using hyphenated cylindrical-square capillaries

Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.     

Analysis of flavonoids by CE using capacitively coupled contactless conductivity detection

A CE method employing capacitively coupled contactless conductivity (C(4)D) compared to indirect UV-detection was developed for the analysis of phytochemically relevant flavonoids, such as 6-hydroxyflavone, biochanin A, hesperetin and naringenin. To ensure fast separation at highest selectivity, sensitivity and peak symmetry, the pH value and the concentration of the running BGE had to be optimized regarding both co- and counter-EOF mode. Optimum conditions were found to be 1.0 and 5.0 mM chromate BGE (pH 9.50) in the counter- and co-EOF mode, respectively. Validation of the established CE-C(4)D method pointed out to be approximately seven times more sensitive compared to indirect UV-detection applying the same conditions. The lower LOD defined at an S/N of 3:1 was found between 0.12 and 0.21 microg/mL for the analytes of interest using C(4)D and between 0.77 and 1.20 microg/mL using indirect UV-detection. Compared to an earlier published CE method employing direct UV-detection, C(4)D was found to be approximately two times more sensitive. Due to the lower baseline noise, C(4)D showed an excellent regression coefficient >0.99 compared to 0.93 when using indirect UV detection calibrating within a concentration range between 1 and 10 microg/mL. The influence of the sugar moiety on the conductivity of a flavonoid was studied upon the analysis of the aglycon hesperetin and the rutinosid hesperidin. The sugar moiety in hesperedin shows a higher conductivity compared to hesperetin. Finally, the optimized established CE-C(4)D method was applied to the determination and quantification of naringenin in Sinupret.     

CE coupling with end-column electrochemiluminescence detection for chiral separation of disopyramide

CE with electrochemiluminescence (ECL) detection technique was successfully applied for the chiral separation of a kind of class IA antiarrhythmic racemic drug. To the best of our knowledge, this is the first report of ECL detection used in chiral CE. To get better detection sensitivity and good enantioresolution at the same time, the conditions of capillary inlet and outlet buffer were systematically optimized. Unlike the traditional chiral separation method, the buffers we used in the capillary inlet and outlet differed from each other in terms of buffer pH, ionic strength, type of BGE as well as buffer composition. Under the optimum conditions, baseline enantioseparation and highly sensitive detection of the enantiomers were achieved. Wide linear relationship of each enantiomer was achieved in the range of 5 x 10(-7) to 2 x 10(-5) mol/L with relative coefficients of 0.996 and 0.997, respectively. The detection limits were estimated to be 8 x 10(-8) and 1.0 x 10(-7) mol/L (S/N = 3) for the enantiomers, respectively. In addition, a successful application of this new method to the chiral separation of the racemic drug in spiked plasma samples confirmed the validity and applicability of the chiral CE-ECL method.     

CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

Identification of polyimide materials using quantitative CE with UV and QTOF‐MS detection

Polyimides (PIs) are a group of widely used synthetic materials that service a variety of different purposes including microelectronics, insulating films and aerospace applications. Depending on the requirements (defined by the particular final product), the actual composition of PIs may show substantial chemical variation. To study this variation in chemical structure, CE‐MS can be employed for the determination of PI composition following chemical degradation of the polymer sample. PI is chemically decomposed to corresponding aromatic diamine and carboxylic acid components using an alkali fusion reaction. Solid polymer samples are fused in a potassium hydroxide melt yielding reaction products that are diluted in acid and can be immediately analysed by CE coupled to a Q/TOF‐MS with quantification performed using conventional UV detection. This approach involves a simple and rapid sample preparation yielding both qualitative and quantitative information regarding the chemical composition of the polymer. Application of the CE‐MS approach is shown for a range of commercially available PI and poly(amide–imide) materials and the results are used to infer the respective chemical compositions.     

Optimization of urinary pteridine analysis conditions by CE‐LIF for clinical use in early cancer detection

The second leading cause of death in the US is cancer and early discovery of the disease has translated into reduced fatality rates. We have identified and performed a systematic investigation of a method for urinary pteridine analysis by using CE‐LIF, which is believed to possess the potential to diagnose the presence of cancer even earlier than existing methodologies. Through system enhancements, we have been able to improve the resolution of the two least resolved sets of peaks (6,7‐dimethylpterin versus 6‐biopterin and D ‐(+)‐neopterin versus 6‐hydroxymethylpterin) from 0.85 to 2.48 and 0.90 to 3.58, respectively. Additionally, we have discovered that the preparation of the urine samples in previous works was inadequate, and we have corrected the method to fully oxidize the pteridines in the urine, resulting in significantly less variability in quantification and greater ease of defining p‐values for healthy versus cancer patients. Finally, we have performed validation steps of spike and recovery and short‐term aging studies to demonstrate the method's robustness. As a result, we present an optimized and validated method ready for transfer from discovery phase to clinical trial that can potentially act as a non‐invasively pre‐screening test for cancer.     

Protein-bound glutathione measurement in cultured cells by CZE with LIF detection

Protein modification due to S-glutathio(ny)lation, usually a reversible process in intact cells, arises interest as a possible mode of regulatory events that may potentially modify a large number of cellular processes. However, since less than 1% of the total protein is S-thiolated in resting cells, high sensitivity methods are required for its evaluation. We set up a new method by CE with LIF detection that allows to measure all forms of intracellular GSH involved in the process. For total and reduced glutathione, cell lysates were rapidly derivatized by 5-iodoacetoamidofluorescein (5-IAF), a selective reagent which traps thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 47 cmx75 microm id capillary by using 7 mmol/L sodium phosphate at pH 11.6. For the evaluation of S-glutathio(ny)lation, intracellular proteins from cell lysates were precipitated and washed to eliminate free GSH. After protein resuspension with NaOH and reduction treatment with tri-n-butylphosphine (TBP), the freed GSH was dried in a vacuum concentrator and directly dissolved in the derivatization mixture. GSH-IAF adduct was detected in a 6 mmol/L sodium phosphate, 3 mmol/L boric acid, and 75 mmol/L N-methylglucamine run buffer in less than 5 min. The high sensitivity ensured by 5-IAF use and sample concentration, allowed to quantify GSH at levels as low as 5 nmol/L, value suitable for the evaluation of protein S-glutathio(ny)lation. The method suitability was checked both in HUVEC and ECV304 cultured cells.     

Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15 degrees C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.     

Determination of medroxyprogesterone acetate residues by CE immunoassay with chemiluminescence detection

A rapid and simple method is developed for the determination of medroxyprogesterone acetate (MPA) by CE immunoassay with chemiluminescence (CL). This method is based on the competitive reactions between horseradish peroxidase (HRP)-labeled MPA (MPA-HRP) and free MPA with anti-MPA antiserum. The influencing factors on the electrophoresis and CL detection were studied completely and the optimal conditions of separation and determination were obtained. The linear range was 2.0-50 nmol/L and the LOD for MPA was 0.9 nmol/L. The present method was applied to the analysis of pork tissues.