Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence

Wise, Dana D.; Shear, Jason B.

doi:10.1016/j.chroma.2005.07.067

Cited by 17 publications

(12 citation statements)

References 26 publications

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“…For each batch of extraction, one flask of cells were digested by 0.05%-tripsin-EDTA (Invitrogen, Carlsbad, CA, USA) and counted with hemacytometer to assist the quantification of compounds per cell. Adapted from extraction methods by Wise and Shear (2006), the metabolites were extracted from the cell monolayer without being digested by tripsin. Briefly, after the medium was removed with pipettes and cotton swabs, the cell monolayer in the flask was rinsed with iced 20 mM glucose in Delbecco's phosphate-buffered saline (DPBS) without calcium and magnesium (Invitrogen, Carlsbad, CA, USA).…”

Section: Introductionmentioning

confidence: 99%

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Silva

Liu

et al. 2008

Biomedical Chromatography

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Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.

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Section: Introductionmentioning

confidence: 99%

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Silva

Liu

et al. 2008

Biomedical Chromatography

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“…MPE native fluorescence has been applied to detect aromatic amino acids (108, 109), neuropeptides (110), catecholamines (111), and hydroxyindoles (112) in CE and leucine aminopeptidase (113) in MCE. …”

Section: Multiphoton Excitation In Native Fluorescence Detectionmentioning

confidence: 99%

Autofluorescence Detection in Analytical Chemistry and Biochemistry

Seeger

2010

Applied Spectroscopy Reviews

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Label-free detection of molecules and particles based on native fluorescence excited at the ultraviolet (UV) region of the electromagnetic spectrum shows great potential for molecular analysis in life sciences. It offers simple, low-cost, and fast methods for sensitive detection of important biological analytes in nature forms. This article reviews the most significant research on label-free native fluorescence detection in analytical chemistry and biochemistry, with particular focus on the instrumental requirements and applications that cover papers published to the beginning of 2009. Future development directions for label-free optical biosensors based on native fluorescence are discussed.

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“…Due to its ultra-low detectability [4], LIF represents one of the most popular detection schemes in CE. Moreover, in combination with some other optical techniques such as polarization [5,6], total internal reflection [7], correlation spectroscopy [8] and multiphoton excitation [9][10][11][12][13], and applications of lowcost light-emitting diode [14][15][16] as well, LIF becomes more attractive, and shows various capabilities to meet the requirements of different samples for analysis.…”

Section: Introductionmentioning

confidence: 99%

On‐column detection of multiphoton‐excited fluorescence in CE using hyphenated cylindrical‐square capillaries

Xu¹,

Chen²,

Feng³

et al. 2008

Electrophoresis

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Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.

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Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence

Cited by 17 publications

References 26 publications

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Autofluorescence Detection in Analytical Chemistry and Biochemistry

On‐column detection of multiphoton‐excited fluorescence in CE using hyphenated cylindrical‐square capillaries

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