FLUORESCENT SUBSTRATES FOR POTENTIAL USE IN ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF MEMBRANE-BOUND NUCLEIC-ACIDS

A number of phosphorylated fluorochromes were synthesized and tested f or the alkaline phosphatase-linked fluorescence assay of membrane-boun d nucleic acids. 3-Hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phos phate ester (HBNP) was found to be the most suitable for the assay. No nfluorescent HBNP is hydrolyzed by phospholipase bound to the probe DN A to the highly fluorescent 3-hydroxy-N,2'-biphenyl-2-naphthalenecarbo xamide (HBN). HBN is insoluble enough in aqueous medium to precipitate on the nylon membrane. Spots containing as little as 5 fg of lambda D NA can be successfully detected on the membrane with HBNP.