title: The use of 3-aminophthalimide as a pro-chemiluminescent label in chemiluminescence and fluorescence-based cellular assays.
abstract: We present a labeling system for direct chemiluminescence-based cellular bioassays using the stable pro-chemiluminescent, luminol precursor, 3-aminophthalimide (API). API-coupled reporter molecules are detected chemiluminometrically after treatment with hydrazine, which converts the API label to luminol. API derivatives containing a variety of functional groups are readily synthesized, allowing for ease of coupling via the imide nitrogen to a host of reporter molecules. The fluorescent nature of APIs further allows for dual fluorescence and chemiluminescence studies. To highlight the utility of this label, we show that API-labeled insulin can be successfully utilized in cellular binding and transport assays and that an API-coupled mitochondrial probe (API-triphenylphosphonium(+)) can be used to both fluorescently and chemiluminometrically investigate mitochondrial function. We also assess the use of API as a polysaccharide and nucleic acid label, and we show that API-labeled palmitic acid undergoes cellular transport and lipid metabolism.
authors.affiliation: Centre for Hepatology at the Royal Free-Hampstead Campus, Royal Free and University College Medical School, London NW32PF, United Kingdom.
authors.forename: Demetra
authors.initials: D
authors.lastname: Mavri-Damelin
authors.forename: Jonathan
authors.initials: J
authors.lastname: Wilden
authors.forename: Ali-Reza
authors.initials: AR
authors.lastname: Mani
authors.forename: Clare
authors.initials: C
authors.lastname: Selden
authors.forename: Humphrey J F
authors.initials: HJ
authors.lastname: Hodgson
authors.forename: Leonard H
authors.initials: LH
authors.lastname: Damelin
mesh_terms: D000818:Animals; D001681:Biological Assay; D045744:Cell Line, Tumor; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D019308:Palmitic Acid / Q000138:chemical synthesis / Q000737:chemistry; D010797:Phthalimides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005452:Fluoresceins; D006834:Hydrazines; D007328:Insulin; D008055:Lipids; C458259:N-aminophthalimide; D009696:Nucleic Acids; D010797:Phthalimides; C029424:hydrazine; D019308:Palmitic Acid; D006493:Heparin