Measurement of oligochitosan–tobacco cell interaction by fluorometric method using europium complexes as fluorescence probes

The interaction of oligochitosan and tobacco cells has been investigated by fluorometric method using two Eu3+ complexes as the probes in this work. Based on the reaction of tobacco cells with oligochitosan conjugated to a strongly fluorescent Eu3+ complex 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl–Eu3+ (oligochitosan–BHHCT–Eu3+ conjugate), the binding kinetic process of oligochitosan–tobacco cells was fluorescently imaged. The results indicate that oligochitosan can be specifically bound to the walls as well as the membranes of tobacco cells. A sensitive and selective Eu3+ complex luminescence probe specific for singlet oxygen, [4′-(10-methyl-9-anthryl)-2,2′:6′,2″-terpyridine-6,6″-diyl]bis(methylenenitrilo)tetrakis (acetate)–Eu3+, was used for developing a new time-resolved fluorescence assay method for the determinations of indole-3-acetic acid (IAA) and peroxidase produced in the cells during the interaction of oligochitosan and tobacco cells. The assays are sensitive with the detection limits of 32 nM for IAA, and 1.2 nM for peroxidase, respectively. The concentration changes of IAA and peroxidase induced by oligochitosan in tobacco cells reveal that oligochitosan can effectively induce the increase of IAA concentration, accompanied by the decrease of peroxidase concentration. These results give a primary and reliable evidence to explain the growth-promoting mechanism of oligochitosan on the plants at molecular level.