Superparamagnetic fluorescent nickel–enzyme nanobioconjugates: synthesis and characterization of a novel multifunctional biological probe

We report, for the first time, the synthesis of superparamagnetic and fluorescent nickel (Ni) nanoparticles (consisting of a single material) directly conjugated to an enzyme, bovine pancreatic α-chymotrypsin (CHT), by chemical reduction in aqueous solution. The structural characterization of Ni–CHT nanobioconjugates was carried out using UV-VIS absorption/photoluminescence spectroscopy and high-resolution transmission electron microscopy. The temperature dependence of the magnetization M(T) taken in zero field cooling and field cooling conditions, exhibits the main features of superparamagnetism. Circular dichroism studies were performed to monitor the structural perturbation to the structure of the enzyme after conjugation with the nickel nanoparticles (Ni NPs). The functional integrity of the enzyme conjugated to the Ni NPs was investigated by monitoring the enzymatic activity of the Ni–CHT conjugates using UV-VIS absorption spectroscopy and comparison with the unbound enzyme under similar experimental conditions. To confirm the conjugation of Ni NPs to CHT, we carried out Forster resonance energy transfer (FRET) studies using a fluorescent probe, 4-nitrophenyl anthranilate (NPA), known to bind at the enzymatic active site of CHT, as the donor (D) and Ni-NP-bound CHT as the acceptor (A). Our studies also demonstrated that the FRET from the donor NPA to the acceptor Ni NPs in CHT can be monitored to follow the D–A distance and hence the protein structure during thermal unfolding. Such a multifunctional superparamagnetic, fluorescent and biologically active nanobioconjugate may be of relevance in nanoparticle-based diagnostic and therapeutic applications.