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

•The F465–Cu2+ ensemble was designed and synthesized for detecting Homocysteine in physiological conditions.•The sensing mechanism that involves F465-Cu2+ based Homocysteine-induced six-membered ring sandwich structure formation was proposed and confirmed.•F465–Cu2+ ensemble was applied in the application of Homocysteine quantification in blood serum.•Evaluating the activities of Cystathionine β-synthase was achieved using F465–Cu2+ ensemble in vitro.

Considering the significant role of plasma homocysteine in physiological processes, two ensembles (F465–Cu2+ and F508–Cu2+) 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) Cu2+ complex. The results of this effort demonstrated that the F465–Cu2+ 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–Cu2+ 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 Cu2+ 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–Cu2+ ensemble for detecting homocysteine in human serum and monitoring the activity of cystathionine β-synthase in vitro.