Low-cost laser-induced fluorescence detector for micellar capillary zone electrophoresis

Zhao, Jianying; Chen, David D. Y.; Dovic̀hi, Norman J.

doi:10.1016/0021-9673(92)87113-m

Cited by 77 publications

(32 citation statements)

References 18 publications

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“…Compared with other amino-labeled fluorescence reagent, such as FITC and TRITC, 13,14 Cy5 showed the higher reaction selectivity and shorter reaction time for primary amines. And the detection limits of Cy5-derivatized amines were the same as those of FITC-derivatized amines.…”

Section: Resultsmentioning

confidence: 98%

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Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Zhang

et al. 2001

ANAL. SCI.

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Noradrenaline and dopamine are two of the most important monoamine neurotransmitters. They play important roles in learning and memory, and in regulation of cardiovascular activity and nervous system. Analysis of the monoamine neurotransmitters has conventionally used high performance liquid chromatography (HPLC) 1-3 or gas chromatography. 4,5 However, the determination of monoamine neurotransmitters in the biological matrix is very difficult because the compounds are usually present at very low concentrations, and many other compounds in biological matrix, which have also absorption in the low-wavelength region, interfere badly with the analysis of monoamine neurotransmitters. Cyanine derivative (Cy5) is a new fluorescence derivative reagent. The excitation and the emission wavelength of Cy5 are 649 and 670 nm, respectively. Thus diode lasers, which emit wavelengths from deep-red to infrared region, are chosen as the light source for laser-induced fluorescence (LIF). This is preferential for determining Cy5-labeled amine neurotransmitters, since almost all of the biological substances have no absorption band in the deep-red and infrared region, resulting in a remarkable decrease of background fluorescence noise and in an increase of detection sensitivity. Recently, LIF is widely used in the analysis of DNA sequence, oligonucleotides and proteins. 6-8But determination of Cy5-labeled monoamine neurotransmitters has not been reported. Capillary electrophoresis (CE) is a newly emerging technique for rapid and high-resolution separation of biomolecules.9 Recently, CE is widely used in the separation and determination of amine. 10 The detection methods of amine in CE include UV-VIS, 11 electrochemical detection 12 and LIF. 13 LIF is one of the most sensitive detection techniques in CE because of its low background signal and the small sample volume. In this paper, we report a rapid, sensitive method for the analysis of noradrenaline and dopamine in CE coupled diode LIF detection, in which Cy5 was chosen as the fluorescence derivative reagent.The effects of buffer concentration, pH value, and reaction time on the derivatization reaction of monoamines were investigated. Experimental ReagentsUnless stated otherwise, all chemicals were of analyticalreagent grade (Beijing Reagent Corp., Beijing, China). Noradrenaline, dopamine and sodium dodecyl sulfate (SDS) were purchased from Sigma (St. Louis, MO, USA). Cy5 was obtained from Amersham Pharmcia Biotech, USA. Water was deionized and purified by a Milli-Q Millipore purification system (Millipore, Marlborough, MA, USA). ApparatusAll analyses were performed on a Beckman P/ACE 5000 system equipped with a Beckman Laser Module 635 nm. This was used as an exciting light source (Beckman-Fullerton, CA, USA). Separations were performed in fused-silica capillaries (57 cm × 75 µm i.d.; Yong Nian Optic Fiber Factory, Hebei Province, China). Derivatization procedureNoradrenaline and dopamine were labeled with Cy5 as follows: Cy5 was dissolved in dimethylformamide. The solution with a...

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Section: Resultsmentioning

confidence: 98%

“…13,14 The result showed the derivatization efficiencies for noradrenaline and dopamine increasing with time; after 90 min, a plateau was reached. Then the derivatization procedure tended to stabilize.…”

Section: Effect Of Reaction Time On Derivatization Reactionmentioning

confidence: 99%

Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Zhang

et al. 2001

ANAL. SCI.

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“…LIF detection is invaluable especially for unparalleled sensitivity [1]; LIF detector combined with a microseparation method can be used to detect extremely low amounts of fluorescent analytes, in some cases at single molecule level [19][20][21]. A number of different LIF systems for miniaturized capillary techniques have been developed.…”

Section: Introductionmentioning

confidence: 99%

Sensitive detection and separation of fluorescent derivatives using capillary electrophoresis with laser-induced fluorescence detection with 532 nm Nd:YAG laser

Vrábel

Táborský

Ryvolová

et al. 2006

Journal of Luminescence

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“…Among these labeling reagents, isothiocyanate (ITC) and succinimidyl ester (SE) derivatives of fluorescein and rhodamine [11,[21][22][23][24] are most commonly used for CE. Though higher sensitivity is attained, the slow reaction kinetics is not favorable for online derivatization required for DCM-CE and other situations where fast labeling reactions are desired.…”

Section: Introductionmentioning

confidence: 99%

Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o‐phthaldialdehyde coupled with fluorescent thiols

Hapuarachchi

Aspinwall

2007

Electrophoresis

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We have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a cross-linking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (epsilon = 78 000 M(-1), quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar(+) laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, we have quantitatively labeled biologically important primary amines in less than 10 s. Detection limits for analysis of glutamate, glycine, GABA, and taurine were less than 2 nM. We present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chemical monitoring of biologically relevant analytes using CE.

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Low-cost laser-induced fluorescence detector for micellar capillary zone electrophoresis

Cited by 77 publications

References 18 publications

Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Sensitive detection and separation of fluorescent derivatives using capillary electrophoresis with laser-induced fluorescence detection with 532 nm Nd:YAG laser

Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o‐phthaldialdehyde coupled with fluorescent thiols

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