Detection of Copper Ion with Laser-Induced Fluorescence in A Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched similar to4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.

Recent advances in the study of biomolecular interactions by capillary electrophoresis

Capillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to 2 review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNA-small molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.

Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. Copyright (C) 2004 John Wiley Sons, Ltd.

Light-emitting-diode-induced fluorescence detector for capillary electrophoresis using optical fiber with spherical end

A simple fluorescence detector for capillary electrophoresis (CE) using a blue light-emitting-diode (LED) as excitation source is constructed and evaluated. An optical fiber was used to collect the fluorescence, and a flat end of the fiber was modified to spherical end, resulting in 50% increase of efficiency over the flat end. A simple device for optical alignment of the fibers and capillary column was designed. The concentration and mass detection limits for fluorescein were 1.8 x 10(-7) Mol l(-1) and 4.3 femol, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site. proved to be the preferential one, being 6.50 x 10(5) 1.94 x 10(6) and 8.94 x 10(5). respectively. In addition. it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions. (C) 2002 Elsevier Science B.V. All rights reserved.

DNA diagnosis by capillary electrophoresis and microfabricated electrophoretic devices

DNA diagnosis is experiencing an impressive progression towards the development of novel technology to identity various clinically relevant categories of genetic changes and to meet the exponential growth of genomics. The introduction of capillary electrophoresis has dramatically accelerated the completion of the first draft of the human DNA sequence in the Human Genome Project, and thus, has become the method of choice for analysis of various genetic variants. The recent development of microfabricated electrophoretic devices has led to the possibility of integrating multiple sample handling with the actual measurement steps required for automation of molecular diagnostics. This review highlights the most recent progress in capillary electrophoresis and electrophoretic microdevices for DNA-based diagnostics, including the important areas of genotyping for point mutation, single nucleotide polymorphisms, short tandem repeats and organism identification. The application of these techniques for infectious and genetic disease diagnosis, as well as forensic identification purpose, are covered. The promising development and the challenges for techinical problems are also discussed.

Quantitative sample injection for capillary electrophoresis

An apparatus including a rotary-type injector was designed for quantitative sample injection in capillary electrophoresis (CE), in which both pressurized flow and electroosmotic flow were used to drive the background electrolyte solution. A relative standard deviation of peak area of lower than 1% was achieved by using this apparatus. The effects of back-pressure regulator, restrictor, and applied voltage on separation efficiency and resolution were investigated. The utility of this apparatus in both micro-HPLC and pressurized capillary electrochromatography (pCEC) was also demonstrated.