BODIPY-based azamacrocyclic ensemble for selective fluorescence detection and quantification of homocysteine in biological applications.

ABSTRACT

Considering the significant role of plasma homocysteine in physiological processes, two ensembles (F465-Cu(2+) and F508-Cu(2+)) were constructed based on a BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) scaffold conjugated with an azamacrocyclic (1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclododecane) Cu(2+) complex. The results of this effort demonstrated that the F465-Cu(2+) ensemble could be employed to detect homocysteine in the presence of other biologically relevant species, including cysteine and glutathione, under physiological conditions with high selectivity and sensitivity in the turn-on fluorescence mode, while the F508-Cu(2+) ensemble showed no fluorescence responses toward biothiols. A possible mechanism for this homocysteine-specific specificity involving the formation of a homocysteine-induced six-membered ring sandwich structure was proposed and confirmed for the first time by time-dependent fluorescence spectra, ESI-MS and EPR. The detection limit of homocysteine in deproteinized human serum was calculated to be 241.4 nM with a linear range of 0-90.0 µM and the detection limit of F465 for Cu(2+) is 74.7 nM with a linear range of 0-6.0 µM (F508, 80.2 nM, 0-7.0 µM). We have demonstrated the application of the F465-Cu(2+) ensemble for detecting homocysteine in human serum and monitoring the activity of cystathionine ß-synthase in vitro.