With an aim to understand the interaction mechanism of bovine serum albumin (BSA) with copper nanoclusters (CuNCs), three different types CuNCs having chemically different surface ligands, namely, tannic acid (TA), chitosan, and cysteine (Cys), have been fabricated, and investigations are carried out in the absence and presence of protein (BSA) at ensemble-averaged and single-molecule levels. The CuNCs, capped with different surface ligands, are consciously chosen so that the role of surface ligands in the overall protein− NCs interactions is clearly understood, but, more importantly, to find whether these CuNCs can interact with protein in a new pathway without forming the "protein corona", which otherwise has been observed in relatively larger nanoparticles when they are exposed to biological fluids. Analysis of the data obtained from fluorescence, ζ-potential, and ITC measurements has clearly indicated that the BSA protein in the presence of CuNCs does not attain the binding stoichiometry (BSA/CuNCs > 1) that is required for the formation of "protein corona". This conclusion is further substantiated by the outcome of the fluorescence correlation spectroscopy (FCS) study. Further analysis of data and thermodynamic calculations have revealed that the surface ligands of the CuNCs play an important role in the protein−NCs binding events, and they can alter the mode and thermodynamics of the process. Specifically, the data have demonstrated that the binding of BSA with TA-CuNCs and Chitosan-CuNCs follows two types of binding modes; however, the same with Cys-CuNCs goes through only one type of binding mode. Circular dichroism (CD) measurements have indicated that the basic structure of BSA remains almost unaltered in the presence of CuNCs. The outcome of the present study is expected to encourage and enable better application of NCs in biological applications.

The present work has been undertaken with an aim to design and develop a material-based nanoscale fluorescence sensory system for the selective and sensitive detection of both highly reactive oxidative species (hROS) and antioxidants in a single chemical entity by exploiting two optically distinguishable useful signals. For this purpose, water-soluble, chitosan-capped fluorescent copper nanoclusters (CuNCs) have been synthesized and characterized by using conventional methods. The sensory action of the CuNCs for several analytes of interest has been investigated at both ensemble-averaged and single-particle levels by exploiting spectroscopic and microscopic techniques. The steady-state fluorescence studies have revealed that CuNCs can act as a very efficient turn-off sensor for hROS (•OH and ClO–) and also a turn-on sensor for antioxidants (ascorbic acid and glutathione). Interestingly, it has been demonstrated that the present sensory system is quite effective in detecting antioxidants in commercial fruit juice and human blood samples at low concentration levels. The feasibility of the hROS sensing ability of CuNCs at single-particle levels is also demonstrated with the help of fluorescence correlation spectroscopy (FCS). Analysis of the data obtained from X-ray photoelectron spectroscopy (XPS) has clearly indicated that the mechanism of turn-off sensing is due to the oxidation of Cu(0)­NCs to Cu­(II) by hROS. On the other hand, fluorescence studies have revealed that the turn-on sensing mechanism is due to the presence of antioxidants, which prevents the oxidation of CuNCs by hROS. More interestingly, investigation has also demonstrated that the optical output signals of the probe–analyte interaction during the hROS/antioxidant signaling can successfully be exploited to construct NAND and IMPLICATION logic gates. Essentially, the outcome of the present investigation demonstrates that CuNCs not only can be effectively used as a nanoscale sensor for both hROS and antioxidants but also have the potential to be used for electronics and medical diagnostics purposes.

Although in recent times nanoparticles (NPs) are being used in various biological applications, their mechanism of binding interactions still remains hazy. Usually, the binding mechanism is perceived to be mediated through either the protein corona (PC) or protein complex (PCx). Herein, we report that the nanoparticle (NP)−protein interaction can also proceed via a different pathway without forming the commonly observed PC or PCx. In the present study, the NP−protein interaction between less-toxic zinc−silver−indium-sulfide (ZAIS) quantum dots (QDs) and bovine serum albumin (BSA) was investigated by employing spectroscopic and microscopic techniques. Although the analyses of data obtained from fluorescence and thermodynamic studies do indicate the binding between QDs and BSA, they do not provide clear experimental evidence in favor of PC or PCx. Quite interestingly, highresolution transmission electron microscopy (HRTEM) studies have shown the formation of a new type of species where BSA protein molecules are adsorbed onto some portion of a QD surface rather than the entire surface. To the best of our knowledge, we believe that this is the first direct experimental evidence in favor of a model-free pathway for NP−protein interaction events. Thus, the outcome of the present study, through experimental evidence, clearly suggests that NP−protein interaction can proceed by following a pathway that is different from classical PC and PCx.