Enzymatic digestion of luminescent albumin-stabilized gold nanoclusters under anaerobic conditions: clues to the quenching mechanism

Abstract

Many of the potential applications of albumin-stabilized gold nanoclusters (AuNCs) arise from the sensitivity of their luminescence to the presence of various ions and albumin-degrading proteases. However, the underlying photophysics and the mechanisms responsible for protease-induced quenching of AuNC luminescence are not fully understood. Here, we study proteinase K-induced digestion of a bovine serum albumin (BSA)–AuNC conjugate under aerobic and anaerobic conditions. To this end, we adapt a Co(II)-catalyzed sulfite-based protocol enabling effective in situ deoxidization without deactivation of the enzyme. In the absence of proteinase K, the anaerobic conditions facilitate the luminescence of BSA–AuNC reflected by a moderate increase in the red luminescence intensity. However, in the presence of proteinase K, we have observed a steep decrease of the emission intensity, irrespective of whether the digestion was carried out under aerobic or anaerobic conditions. In both cases, a decrease of fluorescence occurred “in phase” with shifting of the emission maximum to longer wavelengths. These results contradict the previous hypothesis that protease-induced quenching of BSA–AuNC luminescence is a consequence of enhanced diffusion of oxygen to the bare AuNC. Protease-mediated removal of certain amino acids (e.g. tyrosine) from the vicinity of the AuNCs followed by agglomeration of the AuNCs into non-luminescent assemblies underlies the quenching process. Our findings are discussed in the context of the mechanisms of formation and the photophysics of BSA–AuNC conjugates.