title: Near-infrared fluorescence detection of acetylcholine in aqueous solution using a complex of rhodamine 800 and p-sulfonatocalix[8]arene.
abstract: The complexing properties of p-sulfonatocalix[n]arenes (n = 4: S[4], n = 6: S[6], and n = 8: S[8]) for rhodamine 800 (Rh800) and indocyanine green (ICG) were examined to develop a near-infrared (NIR) fluorescence detection method for acetylcholine (ACh). We found that Rh800 (as a cation) forms an inclusion complex with S[n], while ICG (as a twitter ion) have no binding ability for S[n]. The binding ability of Rh800 to S[n] decreased in the order of S[8] > S[6] >> S[4]. By the formation of the complex between Rh800 and S[8], fluorescence intensity of the Rh800 was significantly decreased. From the fluorescence titration of Rh800 by S[8], stoichiometry of the Rh800-S[8] complex was determined to be 1:1 with a dissociation constant of 2.2 μM in PBS. The addition of ACh to the aqueous solution of the Rh800-S[8] complex caused a fluorescence increase of Rh800, resulting from a competitive replacement of Rh800 by ACh in the complex. From the fluorescence change by the competitive fluorophore replacement, stoichiometry of the Rh800-ACh complex was found to be 1:1 with a dissociation constant of 1.7 mM. The effects of other neurotransmitters on the fluorescence spectra of the Rh800-S[8] complex were examined for dopamine, GABA, glycine, and l-asparatic acid. Among the neurotransmitters examined, fluorescence response of the Rh800-S[8] complex was highly specific to ACh. Rh800-S[8] complexes can be used as a NIR fluorescent probe for the detection of ACh (5 × 10(-4)-10(-3) M) in PBS buffer (pH = 7.2).
authors.affiliation: WPI Immunology Frontier Research Center, Osaka University, Yamada-oka, 1-3, Suita, Osaka 565-0871, Japan. jin@fbs.osaka-u.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Jin
mesh_terms: D000109:Acetylcholine / Q000032:analysis* / Q000737:chemistry; D047250:Calixarenes / Q000737:chemistry*; D004298:Dopamine / Q000737:chemistry; D007208:Indocyanine Green / Q000737:chemistry; D010636:Phenols / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
chemical_list: D010636:Phenols; D012235:Rhodamines; C524897:p-sulfonatocalix(8)arene; C105720:rhodamine 800; D047250:Calixarenes; D005680:gamma-Aminobutyric Acid; D007208:Indocyanine Green; D000109:Acetylcholine; D004298:Dopamine