Fluorescent enzymatic probes suitable for two-photon excitation

Several common aminopeptidase probes are examined for the strength of two-photon excited fluorescence. The fluorophores 4-methoxy-beta-naphthylamine and 7-amino-1-methylcoumarin have limits of detection of 930 pM and 200 pM, respectively. The coumarin derivative is shown to have a two-photon cross section at 590 nm, which is exceptionally large for an aromatic amine. Spectral data for this compound also indicate that excitation near 690 nm should permit quantitation at concentrations as low as 40 pM.