Colloidal semiconductor nanocrystals (SC-NC) present unique and well-controlled physical and chemical properties that make them most suitable, as fluorescent agents, for high resolution imaging and direct follow-up in biological milieus. SC-NC present intense fluorescence that is tunable by size, composition, and shape. In addition, they have large molar extinction coefficients and thermal and photochemical stability, thus avoiding the relatively fast photobleaching characteristic of traditional fluorescent dyes. Nevertheless, constructing SC-NC-labeled, biologically active macromolecules and especially enzymes is challenging. Ensuring the enzyme's stability, sustaining its biological functions, and achieving the desired application all require specific design of the nanoparticles (NPs) properties, their surface coating, and the labeling process. Here, we report an in-depth investigation of the experimental steps involved in the development of a flexible SC-NC labeling toolkit for recombinant human cholinesterases (ChEs), including acetylcholinesterase-R (AChE-R), and the homologous enzyme butyrylcholinesterase (BChE). AChE and BChE, the major acetylcholine (ACh) hydrolyzing enzymes, regulate ACh-mediated neurotransmission, signal transduction, and anti-inflammatory reactions. Impaired ChE functioning is causally involved in many pathologies, including Alzheimer's and Parkinson's diseases, cardiovascular disorders, myasthenia gravis, and Sjogren's syndrome. In addition, exposure to anti-ChE drugs, insecticides, and poisonous nerve agents present prevalent health and security issues. Therefore, recombinant ChEs have been developed both as therapeutic agents and for biomedical research purposes. The labeling toolkit presented here enables optimization of highly emitting SC-NC- ChE conjugates with hydrolytic activity and capacity to bind anti-ChEs, suitable for use for biomedical applications and allows sensitive long-term follow-up of the locations and interactions of these important enzymes in biological systems.
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Acknowledgments The work was supported, in part, by the Israel Ministry of Science and Technology (MOST), Tashtiot program (UB).
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