Fluorescent Substrates for Potential Use in Enzyme-Linked Immunosorbent Assay of Membrane-Bound Nucleic Acids

A number of phosphorylated fluorochromes were synthesized and tested for the alkaline phosphatase-linked fluorescence assay of membrane-bound nucleic acids. 3-Hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HBNP) was found to be the most suitable for the assay. Nonfluorescent HBNP is hydrolyzed by phospholipase bound to the probe DNA to the highly fluorescent 3-hydroxy-N,2'-biphenyl-2-naphthalenecarboxamide (HBN). HBN is insoluble enough in aqueous medium to precipitate on the nylon membrane. Spots containing as little as 5 fg of lambda DNA can be successfully detected on the membrane with HBNP.