Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-Induced Fluorescence

Cheng, Yung‐Fong; Dovic̀hi, Norman J.

doi:10.1126/science.3140381

Cited by 537 publications

(261 citation statements)

References 21 publications

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“…In this case, the electroosmotic flow is suppressed and the ionic species are separated by seiving effect. CE has been coupled to different detection methods including ultraviolet UV-visible absorption [11], conductimetry, mass spectrometry (MS), patch clamp, electrochemical (EC) [4,13] or LIFD [14,15]. This versatility of separation and detection methods has allowed analyzing a wide spectrum of substances ranging from ions to proteins.…”

Section: Capillary Electrophoresis (Ce)mentioning

confidence: 99%

“…They obtained a limit of concentration detection at 10 À10 M level. The introduction of post-column detection in a sheat flow cuvette [14] or collinear geometry [26] lowered the limit of concentration detection to 10 À13 M. These improvements made single molecule detection the ultimate level of precision for CZE analysis [15,[27][28][29][30]. As a consequence, the CE high resolution power and sensitivity within the zeptomolar range (10 À21 M) made possible the successful use of CE-LIFD in endogenous neuroactive substances, DNA and illicit drug analysis [5,6,31].…”

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

“…The peaks of radiation absorption of the fluorophore should match the wavelength of the laser in order to reach maximum sensitivity. Thus, LIFD requires a simple sample pre-treatment with a dye to make the analytes fluorescent in order to detect them [14,15,29].…”

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

“…It is necessary to have at least 16-18 h of incubation to obtain stable and well detected FITC derivatives [29,30]. CE-LIFD analysis of FITC derivatives gives subattomolar determination of AA mixture, which represents few thousands of injected molecules [14,27,29,39,40].…”

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

“…The combination of CZE high efficient separation and LIFD high sensitivity detection, in a fused silica capillary filled with alcaline carbonate buffer, is especially good for acidic AA such as glutamate (Glu) and aspartate (Asp), and for basic AA such as arginine (Arg) and lysine (Lys). The detection limit reaches the subattomol level [14,15,26,29,30,116]. The applicability of CE may be enhanced if the spectral analysis is incorporated.…”

Section: Ce-lifd Coupled To Microdialysis Studies Neuroactive Substamentioning

confidence: 99%

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Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Páez

Hernández

2001

Biopharm & Drug Disp

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Capillary electrophoresis (CE) is a high-efficiency analytical technique that has had a great impact as a tool in biomedical research, clinical and forensic practice in the last ten years. Only in one of the applications, the DNA analysis, it has had an explosive exponential growth in the last few years. This impact is expressed in an enormous amount of CE articles and many reviews. The CE advantages with respect to other analytical techniques: the required very small sample volume, rapid analysis, great resolution power and low costs, have made this technique ideal for the analysis of a numerous endogenous and exogenous substances present in biological fluids. The different modes of CE have been coupled to different detection techniques such as UV-absorbance, electrochemical, mass spectrometry and laser-induced fluorescence detection (LIFD) to detect different nature and molecular size separated analytes. This review focuses mostly on the applications of CE-LIFD, to measure drugs and endogenous neuroactive substances such as amino acids and monoamines, especially in microdialysis samples from experimental animals and humans. CE-LIFD trends are discussed: automated faster analysis with capillary array systems, resolution power improvement, higher detection sensitivity, and CE systems miniaturization for extremely small sample volume, in order to make CE easier and affordable to the lab bench or the clinical bed.

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Section: Capillary Electrophoresis (Ce)mentioning

confidence: 99%

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

Section: Ce-lifd Coupled To Microdialysis Studies Neuroactive Substamentioning

confidence: 99%

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Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Páez

Hernández

2001

Biopharm & Drug Disp

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Characterization of fluorescein–oligonucleotide conjugates and measurement of local electrostatic potential

1998

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The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono‐ and dianion, and the pKa relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single‐ and double‐stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to −28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein–oligonucleotide conjugates can be calculated by assuming: ε494 = 62000/[1 + 10−(pH−6.90)] + 12000/[1 + 10(pH−6.90)] (M−1 cm−1). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation. © 1998 John Wiley & Sons, Inc. Biopoly 46: 445–453, 1998

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Correlating cell cycle with metabolism in single cells: Combination of image and metabolic cytometry

et al. 1999

Self Cite

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Background:We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. Methods: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. Results: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G 1 phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G 2 /M cells. Conclusions: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle. Cytometry 37: 14-20, 1999. 1999

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Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-Induced Fluorescence

Cited by 537 publications

References 21 publications

Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Characterization of fluorescein–oligonucleotide conjugates and measurement of local electrostatic potential

Correlating cell cycle with metabolism in single cells: Combination of image and metabolic cytometry

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