Fu-Tai A. Chen

Rapid protein analysis by capillary electrophoresis

Abstract Rapid protein analysis by free-zone capillary electrophoresis in an untreated fused-silica column is presented. Separation of model proteins by the proposed method shows that the retention times of these proteins correlate well with each of their isoelectric points. By using a high voltage gradient, most proteins could be separated in 200 s with excellent reproducibility. Similarly, human serum proteins could be resolved and determined in less than 100 s.

Characterization of highly sulfated cyclodextrins.

A class of highly sulfated cyclodextrins (HS-CDs) was developed for enantiomeric separation of chiral compounds by capillary electrophoresis (CE). The HS-CDs were produced by a facile single-step direct sulfation of cyclodextrin using sulfur trioxide-trimethylamine complex in dimethylformamide. Characterization of the HS-CDs by electrospray ionization mass spectrometry and by CE using a well-established indirect detection method indicated the species have very narrow heterogeneity in terms of degree of sulfation. Elemental analysis of the HS-alpha-, beta- and gamma-CDs showed that the average sulfate contents were 11, 12, and 13 per CD molecule, respectively. The 13C NMR of HS-CDs is consistent with the structural assignment of nearly complete sulfation at C-6 primary hydroxyl groups and partial sulfation at the C-2 secondary hydroxyls (>70%), while the C-3 hydroxyls remain unsubstituted. Enantiomeric separation by CE using the HS-CDs as chiral selectors showed that HS-alpha-, beta- and gamma-CDs complement each other by exhibiting different chiral selectivities, resulting in resolution of many chiral neutral, acidic and basic compounds of greatly varying structural features. The part of HS-CD that interacts with the guest molecule during complexation and, therefore, the receiving end of the cyclodextrin hydrophobic bucket was surrounded with largely regiospecifically substituted C-2 sulfates and intact C-3 hydroxyls, both at the equatorial positions. Such global regiospecific structural arrangement in HS-CDs provides differential diasteroisomeric complexation is proposed to be the principal contributing factor in the resolving racemates.

Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence Application to homogeneous digoxin immunoassay

A laser-induced fluorescence (LIF) immunoassay technique based on capillary electrophoretic (CE) separation is demonstrated. The analysis of digoxin in serum at clinically useful concentration levels of 10(-9) to 10(-10) M is achieved using this technique. The chemistry presented here using digoxigenin-labeled B-phycoerythrin was selected as a convenient model for the exploration of CE-LIF-based immunoassays. The LIF system described here exhibits detection limits in the low 10(-11) M range for several common fluorophores. The data presented in this report are one of the first examples of nanomolar quantitative analysis in a human serum matrix by CE.

Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye

Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicarboxylic acid. A Cy5-labeled oligonucleotide M13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2.5-mW red semiconductor laser is about 10(-10) M.

Characterization of charge-modified and fluorescein-labeled antibody by capillary electrophoresis using laser-induced fluorescence Application to immunoassay of low level immunoglobulin A

Abstract An affinity-purified and fluorescein-labeled antibody was chemically modified to alter its electrophoretic mobility. The functional activity of the resulting modified and fluor labeled antibody appeared was preserved. Immunoassay is carried out by the addition of an appropriate amount of the labeled antibody to a sample containing the antigen. Separation of the antigen-bound and free labeled antibody was performed by capillary electrophoresis with laser- induced fluorescence detection. Both free and antigen-bound labeled antibody species may be analysed simultaneously. Quantitation of the immunoassay is achieved with an on-line data analysis.

Characterization of food proteins by capillary electrophoresis

Abstract A simple analytical method for characterization of food proteins by capillary electrophoresis (CE) has been developed. Major proteins in chicken eggs and cows milk are characterized and can be quantitated by the CE technique. Egg white proteins are well resolved while the yolk shows a substantially more complex protein separation pattern than that in the egg white. Caseins and whey proteins in fresh milk were resolved by CE in a similar buffer system. Separation of both milk and egg proteins was performed reliably and reproducibly in an untreated fused-silica column, 25 cm × 20 μm I.D. Diluted egg or milk samples can be directly loaded on an automated instrument with on-line injection, detection and real-time data analysis in less than 10 min.

Characterization of TO-PRO-3 as an intercalator for double-stranded DNA analysis with red diode laser-induced fluorescence detection

Abstract We report the analysis of double-stranded DNA (dsDNA) with a blue intercalating dye, TO-PRO-3 (TP3) using a visible diode laser-induced fluorescence (LIF) detection. In the presence of single-stranded DNA (ssDNA), such as pd(A) 40–60 or pd(T) 20–40 , TP3 adsorption maximum shifts slightly from 631 to 633 nm and, when intercalated to dsDNA, the absorption maxima shifts to 643 nm. TP3 itself does not fluoresce in the presence of eithet d(A)_ 18 or d(T) 18 alone, but the combination of both oliginucleotide species yielded intense fluorescence, about two orders of magnitude higher. The LIF detection was based on the use of a 2.5-mW diode laser emitting light at 635 nm and the fluorescence of the resulting dsDNA-bound TP3 was collected at 670 nm. The capillary electrophoretic (CE) separation of fragments from 72 to 1353 basepairs (bp) of ΦX174/ Hae III digest were well-resolved within 10 min in the gel-buffer system with TP3. The application of TP3 as an intercalating dye for the analysis of dsDNA fragments produced by polymerase chain reaction (PCR) was examined. Excellent correlations between CE-LIF area and fragment size in basepairs were obtained with TP3 and ethidium bromide as the intercalating dyes. TP3-based chemistry along with the diode laser indiced fluorescence detection system is well-suited for rapid, high-sensitivity and automated DNA analysis of the PCR-amplified dsDNA products and DNA restriction fragments.

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection.

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.

Detection of amplified Y chromosome-specific sequence by capillary electrophoresis with laser-induced fluorescence**Supported by Advanced Technology Center, Beckman Instruments Inc., Fullerton, California.

OBJECTIVE To develop a sensitive method for genetic diagnosis using capillary electrophoresis with laser-induced fluorescence. DESIGN Using male DNA diluted with female DNA as an example, ZFY gene from Y chromosome was amplified specifically by polymerase chain reaction (PCR) with fluorescence-labeled primers and detected by capillary electrophoresis with laser-induced fluorescence. SETTING Laser operating laboratory. PARTICIPANTS Human male and female DNA were extracted from healthy human male and female subjects. INTERVENTIONS None. MAIN OUTCOME MEASURES The concentration of human DNA was determined by using a spectrophotometer at an absorbance of 260 nm. RESULT Deoxyribonucleic acid fragments amplified from a single copy of ZFY gene were detected by capillary electrophoresis with laser-induced fluorescence after 35 cycles of PCR amplification. CONCLUSION This method is potentially applicable for rapid and sensitive detection of fetal Y chromosome DNA sequence in maternal circulation and of single-cell DNA diagnosis.