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.

Capillary array electrophoresis with confocal fluorescence rotary scanner

A capillary array electrophoresis system with rotary corifocal fluorescence scanner was reported. High speed direct current rotary motor combined with a rotary encoder and the reflection mirror has been designed to direct exactly the excitation laser beam. to the array of capillaries, which are symmetrically distributed around the motor. The rotary encoder is introduced to accurately orient the position of each capillary and its output signal triggers the data acquiring system to record. the fluorescence signal corresponding to each capillary. Separations of several amino acids are demonstrated by eight-channel capillary array electrophoresis built by ourselves.

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.

Capillary electrophoretic analysis of arsenic species with indirect laser induced fluorescence detection

The development of a method for determining arsenic species by capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence (LIF) is described in this paper. The buffer pH, the concentration of fluorescein, the nature and the concentration of the background electrolytes (BGEs) were defined. When 2.0 mM NaHCO3 (pH 9.28) with 10(-7) M fluorescein was used as the buffer, arsenite (As(lll), dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), and arsenate (As(V)) were all separated from one another. The limits of detection for the four arsenic species were p p in the range of 0.12-0.54 mg/L. This method was used in the analysis of spiked arsenic species in tap and mineral water to demonstrate its usefulness. The results showed that both the recovery and the reproducibility of the developed method were acceptable.

The application of spline wavelet transform to the determination of electroosmotic mobility in capillary electrophoresis

The rule of current change was studied during capillary electrophoresis (CE) separation process while the conductivity of the sample solution was different from that of the buffer. Using a quadratic spline wavelet of compact support, the wavelet transforms (WTs) of capillary electrophoretic currents were performed. The time corresponding to the maximum of WT coefficients was chosen as the time of current inflection to calculate electroosmotic mobility. The proposed method was suitable for different CE modes, including capillary zone electrophoresis, nonaqueous CE and micellar electrokinctic chromatography. Compared with the neutral marker method, the relative errors of the developed method for the determination of electroosmotic mobility were all below 2.5%. (C) 2002 Elsevier Science B.V. All rights reserved.

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.

Evaluation of the interaction between netropsin and double stranded DNA by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to study the interaction between netropsin and a 14mer double stranded DNA (dsDNA). The binding constant of this interaction calculated from Scatchard plot was (1.07+/-0.10) X 10(5) (mol/L)(-1). The binding stoichiometry was 1:1. The use of polyacrylamide coated capillary showed better effect in the analysis of DNA than noncoated capillary.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH4HF2. The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50degreesC, even 20 fmol cytochrome c could be well digested and detected.