Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection

Wang, Chunling; Zhao, Shulin; Yuan, Huatang; Xiao, Dingquan

doi:10.1016/j.jchromb.2006.01.013

Cited by 45 publications

(26 citation statements)

References 40 publications

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“…16 However, the addition of fluorescent labels to proteins can affect their ligand binding, solubility and stability, and it is also difficult to ensure the attachment of only a single label per protein molecule. 17,18 The measurement of intrinsic protein fluorescence has also been demonstrated previously in microchannels for the label-free detection of proteins and their conformational changes, by using lasers [19][20][21] or light-emitting diodes (LED's) 22,23 for the excitation of samples. However, these techniques have not yet been sufficiently accurate to obtain thermodynamic parameters such as the free-energy of protein unfolding, as is required for use in drug-discovery, formulation or protein engineering applications.…”

Section: Introductionmentioning

confidence: 97%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

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Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10 8 molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

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Section: Introductionmentioning

confidence: 97%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

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“…Detection of NDA derivatives has commonly relied on HeCd or Ar 1 lasers with low nM detection limits for proteins and amino acids [13]. Recently, a blue LED was used to detect NDA-labeled amino acids in cerebrospinal fluids with low nM (10-30 nM) detection limits [14,15]. Xiao and co-workers also reported analysis of amino acids using LED-IF with low nM sensitivity [16].…”

Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis with a UV light‐emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

2006

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We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.

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“…A CE optical fiber LED-induced fluorescence detection system was developed for the analysis of the excitatory amino acids (EAAs) tagged with NDA [42]. The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm675 mm id) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV.…”

Section: Analytical Applicationsmentioning

confidence: 99%

CE detector based on light‐emitting diodes

et al. 2007

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CE detectors based on light-emitting diodes (LEDs) as light sources are receiving considerable attention due to their exceptionally high stability, high intensity, low cost, and a variety of wavelengths in the UV and visible spectrum. This article is a comprehensive review on CE methods using LED-based detectors with absorbance and fluorescence detection, and several applications on the determination of riboflavin, bacteria, drug, and amino acids in biological samples are described.

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Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection

Cited by 45 publications

References 40 publications

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Capillary electrophoresis with a UV light‐emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

CE detector based on light‐emitting diodes

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