Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols.
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.
Hapuarachchi
Suminda
S
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
Aspinwall
Craig A
CA
D000596:Amino Acids / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry*; D018377:Neurotransmitter Agents / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D009764:o-Phthalaldehyde / Q000737:chemistry*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D018377:Neurotransmitter Agents; D013438:Sulfhydryl Compounds; D009764:o-Phthalaldehyde