The use of 3-aminophthalimide as a pro-chemiluminescent label in chemiluminescence and fluorescence-based cellular assays.

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.