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

Chen, Fu-Tai A.; Tusak, Anton; Pentoney, Stephen L.; Konrad, K.; Lew, Clarence; Koh, E.; Sternberg, James C.

doi:10.1016/0021-9673(93)83253-o

Cited by 36 publications

(17 citation statements)

References 5 publications

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“…Recently, LIF is widely used in the analysis of DNA sequence, oligonucleotides and proteins. [6][7][8] But 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.…”

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confidence: 99%

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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confidence: 99%

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

Zhang

et al. 2001

ANAL. SCI.

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“…However, all of these modifications required specialized technology for their production, some of which is beyond the capabilities of most laboratories. A simpler approach is the incorporation of laser-induced fluorescence (LIF) detectors using diode lasers (40)(41)(42). The development of the cyanine dyes (Cy3, Cy5), capable of labelling primary amino groups via reactive succinimide ester groups, has greatly enhanced not only labelling efficiency but also signal output (19,(42)(43)(44).…”

Section: Discussionmentioning

confidence: 99%

Analysis of single‐cell cultures by immunoaffinity capillary electrophoresis with laser‐induced fluorescence detection

Phillips

2001

Luminescence

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Neuropeptide regulation of immunological activity is becoming an important issue in both basic and clinical sciences, necessitating the need for analysis to be performed at the single-cell level. A microsampling procedure has been developed for studying secretion of biologically important peptides from neuropeptide-stimulated lymphocytes, based on microdialysis sampling coupled to immunoaffinity capillary electrophoresis (ICE), with laser-induced fluorescence (LIF) detection using a fibre-optic spectrometer and diode laser excitation. The system demonstrated a limit of detection in the high attomole (10(-18) mol/L) range with pure standards and was capable of monitoring secretion from a single cell over time. Using this system it was possible to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines. CD4(+) lymphocytes demonstrated a 7.5-fold increase in cytokine secretion over baseline following stimulation with substance P (SP) and calcitonin gene-related peptide (CGRP). B cells responded to CGRP and vasoactive intestinal peptide (VIP) stimulation (5.5-fold increase), but not to SP. These changes took place 12--20 h post-stimulation and, once the peak secretion had been reached, remained at that level for the duration of the experiment. This system demonstrates the ability to perform high sensitivity measurements on microsamples of biological fluids.

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“…Cy5 has two broad absorbtion maxima at l ex,1 = 630 nm (e 1 = 150 000 M ±1 cm ±1 ) and l ex,2 = 655 nm (e 2 = 215 000 M ±1 cm ±1 ), and a fluorescence emission maximum at l em = 6691 nm [18,19]. Cy5.5 has an absorbtion maximum at l ex = 675 nm (e = 200 000 M ±1 cm ±1 ), and a fluorescence emission maximum at l em = 696 nm [20,21].…”

Section: Fluorescent Dyesmentioning

confidence: 99%

“…In the latter strategy, a primer is made initially with an N-amino group, which is then coupled to an activated form of Cy5 or Cy5.5 dye. General procedures for coupling of cyanine-type dyes to N-aminomodified oligodeoxynucleotides have been described [19,27,28].…”

Section: Terminators and Primersmentioning

confidence: 99%

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer

Yager

Baron²,

Batra³

et al. 1999

Electrophoresis

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The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 μm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far‐red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle‐sequencing of an M13 single‐stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse‐transcribed human immunodeficiency virus (HIV‐1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence‐determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 μm thick MicroCel gels. We routinely obtain well‐resolved sequence which can be base‐called with 98.5% accuracy to position ˜450 on an 11 cm gel, and to position ˜900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution‐limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye‐labeled primers and a DNA polymerase of the F667Y, Δ(5′ → 3′ exo) class, we can routinely detect single‐nucleotide mutations and polymorphisms over the 0.35—0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV‐1 RNA isolate.

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

Cited by 36 publications

References 5 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

Analysis of single‐cell cultures by immunoaffinity capillary electrophoresis with laser‐induced fluorescence detection

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer

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