Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly(methyl methacrylate) microchips

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65 degreesC). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated microsystem, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/muL could be finished within 15 min, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection.

Abnormal phenomenon of the dependence of the retention factors for uncharged species on applied voltage in capillary electrochromatography

Polymethacrylate-based monolithic columns were prepared for capillary electrochromatography (CEC) by in situ copolymerization of butyl methacrylate (BMA), 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and ethylene dimethacrylate (EDMA) in the presence of a porogen in fused-silica capillaries of 100 mum I.D. The abnormal phenomenon that retention factors for neutral species decreases with applied voltage in CEC was observed. Capillary electrophoresis (CE) instruments usually require a period of time to increase voltage from 0 kV to desired value, which is called as ramp time. Such ramp time and any error in the determination of dead time should be taken into account during the accurate calculation of retention factors. After the correction of the retention factors, the plots of the corrected factors for alkylbenzene versus applied voltage were made, the absolute value of the plot slopes are less than 1.8 X 10(-4), Which indicates that the corrected retention times for neutral species do not show any dependence on applied voltage. Further, the plots of the corrected retention times for acidic and basic compounds versus the reciprocal of applied voltage were drawn, where the target compounds were eluted in neutral form. The very nice linearity of the plots was obtained. The linear correlation coefficients are over 0.999. Here, the slopes of the plots represent

Reversed capillary electrochromatography: Investigation of peak symmetry for eluting curves of solutes

The transport processes of components in capillary electrochromatographic column was investigated based on the basic model of relaxation theory. A principal transport equation of chromatographic relaxation theory was established and mathematical expressions for eluting curves were obtained under the situations of both capillary electrophoresis and chromatography. Characteristics of peak symmetry and its effecting factors are discussed. Tailing peaks, symmetrical peaks and fronting peaks would be observed simultaneously, which was further proved with reversed capillary electrochromatographic experiments.

Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresis can be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated. (C) 2002 Elsevier Science B.V. All rights reserved.

Pressurized capillary electrochromatography separation of peptides with strong cation exchange and hydrophilic interaction

A pressurized capillary electrochromatography (pCEC) instrument with solvent gradient capability has been used for the separation of a peptide mixture. Retention mechanism and selectivity of the peptides were studied by pCEC using a strong cation exchange (SCX) column. The effects of applied voltage, supplementary pressure, organic modifier concentration, ionic strength,, and pH value on pCEC separation were investigated. It was found that the retention mechanism of the peptides in this system is based on a mixed mode of hydrophilic interaction, strong cation exchange, and electrophoresis. Compared with the separation results obtained by reverse phase pCEC and capillary electrophoresis (CE), this mixed-mode pCEC is more powerful for the separation of hydrophilic peptides with similar charge-to-mass ratio.

Capillary electrophoretic study on UDP-sugars in cells

Glucose is an important regulator of cell growth and metabolism. Uridine diphosphate sugars (UDP-sugars), as the intermediate products of metabolism, play pivotal roles as precursors in the synthesis of complex carbohydrates and glycolipids as well as lectose. It is very important to study their metabolism in cells in clinical biochemistry. A capillary electrophoretic method has been developed for the analysis of UDP-sugars and nucleotides, By using an uncoated capillary (70cm x 50 mu m) and 20 mmol/L borax buffer (pH 9), 4 important UDP-sugars can be analyzed in 15 min at 22 kV with satisfactory precision and sensitivity. The developed method has been applied to analyze UDP-sugars concentrations in lymphocytes, fibroblasts and mesangial cells, and the results show it not only is much better than HPLC method, but also can be used to measure the energy charge of cells.

Protein A immobilized monolithic capillary column for affinity chromatography

Monolithic capillary columns for affinity chromatography were prepared by an in situ polymerization procedure using glycidyl methacrylate (GMA) as a monomer and trimethylolpropane trimethacrylate (TRIM) and ethylene dimethacrylate (EDMA) as cross-linkers, respectively. Scanning electron microscopy was applied to characterize the morphology of the end of monolithic capillary and mercury intrusion porosimetry to characterize the polymer rod prepared within the confines of a stainless steel column with 50 mm x 4.6 mm i.d. under the same polymerization condition. Obvious differences in the porous properties between the TRIM- and EDMA-based monoliths could be observed. Moreover, the mechanical stability of these two monolithic capillary columns was compared by testing the reproducibility of the column performance. The rod prepared with GMA and TRIM proved to be mechanically more stable than that prepared with GMA and EDMA. Protein A was immobilized on the monolithic rod for affinity chromatography and the experiments were performed on a capillary electrophoresis instrument, using its pressure system as the driving force. Non-specific adsorption was not observed on the TRIM-based affinity column, as proved with bovine serum albumin (BSA) as a test protein. The affinity column prepared with GMA and TRIM was then applied to determine the hIgG concentration in human serum. The correlative coefficient of the calibration curve reached 0.9942. The amount of adsorbed hIgG was unaffected by the flow rate of the loading buffer, which makes this method suitable for fast determination of biomacromolecules in microliter samples. (C) 2002 Elsevier Science B.V All rights reserved.

Luminescent supramolecular microstructures containing Ru(bpy)(3)(2+): Solution-based self-assembly preparation and solid-state electrochemiluminescence detection application

In this correspondence, we report on the first preparation of novel, robust Ru(bpy)(3)(2+)-containing supramolecular microstructures via a solution-based self-assembly strategy, carried out by directly mixing H2PtCl6 and Ru(bpy)(3)Cl-2 aqueous solutions at room temperature. It reveals that both the molar ratio and concentration of reactants have a heavy influence on the morphologies of such microstructures. The electrochemical behavior of the Ru(bpy)(3)(2+) components contained in the solid film of the microstructures formed on the electrode surface is also studied and found to exhibit a diffusion-controlled voltammetric feature. Most importantly, such microstructures exhibit excellent electrochemiluminescence (ECL) behaviors and therefore hold great promise as new luminescent materials for solid-state ECL detection in capillary electrophoresis (CE) or CE microchip.

Analysis of five pharmacologically active compounds from the Tibetan medicine Elsholtzia with micellar electrokinetic capillary chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20 degrees C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5 x 10(-4)similar to 0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1 x 10(-3) similar to 0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were ( 99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N = 3) were 1.1 x 10(-4) mg/mL for quercetin, 2.6 x 10(-4) mg/mL for rutin, 1.8 x 10(-4) mg/mL for saussurenoside, 2.9 x 10(-4) mg/mL for kaempferol, and 6.3 x 10(-4) mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.

Capillary electrophoretic analysis of pharmacologically active xanthone compounds from Swertia przewalskii pissjauk extract

Pharmacologically active xanthone compounds isolated from Swertia przewalskii pissjauk were well separated by capillary electrophoresis (CE) within 5 min, using a running buffer of 25 mM disodium tetraborate at pH 9.0. Quantitative determination was shown to be possible because regression equations revealed a linear relationship between the peak area of each constituent and its concentration, with correlation coefficients of 0.9972-0.9994. The relative standard deviations were between 0.44%-0.73% for migration times and 2.52%-4.28% for peak areas. The dissociation constant of 1,7-O-beta-D-glucopyranosyl-8-hydroxy-3,7-dimethoxyxanthone, 1,8-dihydroxy-3, 7-dimethoxy-xanthone and 1,7-dihydroxy-3,8-dimethoxyxanthone were also measured by the CE method, giving a value of 9.04, 8.94, and 8.59, respectively.

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240 000 to 460 000/m, and the relative standard deviation for t(0) and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated. (C) 2000 Elsevier Science B.V. All rights reserved.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

Development of low-cost sensing and separation devices based on macro, micro and nano technology for health applications

The work presented in this thesis described the development of low-cost sensing and separation devices with electrochemical detections for health applications. This research employs macro, micro and nano technology. The first sensing device developed was a tonerbased micro-device. The initial development of microfluidic devices was based on glass or quartz devices that are often expensive to fabricate; however, the introduction of new types of materials, such as plastics, offered a new way for fast prototyping and the development of disposable devices. One such microfluidic device is based on the lamination of laser-printed polyester films using a computer, printer and laminator. The resulting toner-based microchips demonstrated a potential viability for chemical assays, coupled with several detection methods, particularly Chip-Electrophoresis-Chemiluminescence (CE-CL) detection which has never been reported in the literature. Following on from the toner-based microchip, a three-electrode micro-configuration was developed on acetate substrate. This is the first time that a micro-electrode configuration made from gold; silver and platinum have been fabricated onto acetate by means of patterning and deposition techniques using the central fabrication facilities in Tyndall National Institute. These electrodes have been designed to facilitate the integration of a 3- electrode configuration as part of the fabrication process. Since the electrodes are on acetate the dicing step can automatically be eliminated. The stability of these sensors has been investigated using electrochemical techniques with excellent outcomes. Following on from the generalised testing of the electrodes these sensors were then coupled with capillary electrophoresis. The final sensing devices were on a macro scale and involved the modifications of screenprinted electrodes. Screen-printed electrodes (SPE) are generally seen to be far less sensitive than the more expensive electrodes including the gold, boron-doped diamond and glassy carbon electrodes. To enhance the sensitivity of these electrodes they were treated with metal nano-particles, gold and palladium. Following on from this, another modification was introduced. The carbonaceous material carbon monolith was drop-cast onto the SPE and then the metal nano-particles were electrodeposited onto the monolith material

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Versatile microanalytical system with porous polypropylene capillary membrane for calibration gas generation and trace gaseous pollutants sampling applied to the analysis of formaldehyde, formic acid, acetic acid and ammonia in outdoor air

The analytical determination of atmospheric pollutants still presents challenges due to the low-level concentrations (frequently in the mu g m(-3) range) and their variations with sampling site and time In this work a capillary membrane diffusion scrubber (CMDS) was scaled down to match with capillary electrophoresis (CE) a quick separation technique that requires nothing more than some nanoliters of sample and when combined with capacitively coupled contactless conductometric detection (C(4)D) is particularly favorable for ionic species that do not absorb in the UV-vis region like the target analytes formaldehyde formic acid acetic acid and ammonium The CMDS was coaxially assembled inside a PTFE tube and fed with acceptor phase (deionized water for species with a high Henry s constant such as formaldehyde and carboxylic acids or acidic solution for ammonia sampling with equilibrium displacement to the non-volatile ammonium ion) at a low flow rate (8 3 nLs(-1)) while the sample was aspirated through the annular gap of the concentric tubes at 25 mLs(-1) A second unit in all similar to the CMDS was operated as a capillary membrane diffusion emitter (CMDE) generating a gas flow with know concentrations of ammonia for the evaluation of the CMDS The fluids of the system were driven with inexpensive aquarium air pumps and the collected samples were stored in vials cooled by a Peltier element Complete protocols were developed for the analysis in air of NH(3) CH(3)COOH HCOOH and with a derivatization setup CH(2)O by associating the CMDS collection with the determination by CE-C(4)D The ammonia concentrations obtained by electrophoresis were checked against the reference spectrophotometric method based on Berthelot s reaction Sensitivity enhancements of this reference method were achieved by using a modified Berthelot reaction solenoid micro-pumps for liquid propulsion and a long optical path cell based on a liquid core waveguide (LCW) All techniques and methods of this work are in line with the green analytical chemistry trends (C) 2010 Elsevier B V All rights reserved