2. Academic Validation
  3. Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence

Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence

  • J Anal Methods Chem. 2015;2015:368362. doi: 10.1155/2015/368362.
Christian G Kendall 1,  Amanda M Stockton 2,  Stephen Leicht 3,  Heather McCaig 4,  Shirley Chung 4,  Valerie Scott 4,  Fang Zhong 4,  Ying Lin 4
Affiliations

Affiliations

  • 1 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA ; Weill Cornell Graduate School of Medical Science, New York, NY 10065, USA.
  • 2 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA ; Georgia Institute of Technology, Atlanta, GA 30332, USA.
  • 3 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA ; University of California, Los Angeles, CA 90095, USA.
  • 4 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA.
PMID: 26090268 DOI: 10.1155/2015/368362
Abstract

Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all Solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5-17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.

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