title: Fluorescein-5-isothiocyanate-conjugated protein-directed synthesis of gold nanoclusters for fluorescent ratiometric sensing of an enzyme-substrate system.
abstract: This study describes the synthesis of a dual emission probe for the fluorescent ratiometric sensing of hydrogen peroxide (H2O2), enzyme activity, and environmental pH change. Green-emitting fluorescein-5-isothiocyanate (FITC) was conjugated to the amino groups of bovine serum albumin (BSA). This FITC-conjugated BSA acted as a template for the synthesis of red-emitting gold nanoclusters (AuNCs) under alkaline conditions. Under single wavelength excitation, FITC/BSA-stabilized AuNCs (FITC/BSA-AuNCs) emitted fluorescence at 525 and 670nm, which are sensitive to changes in solution pH and H2O2 concentration, respectively. The effective fluorescence quenching of AuNCs by H2O2 enabled FITC/BSA-AuNCs to ratiometrically detect the H2O2 product-related enzyme system and its inhibition, including glucose oxidase-catalyzed oxidation of glucose, acetylcholinesterase/choline oxidase-mediated hydrolysis and oxidation of acetylcholine, and paraoxon-induced inhibition of acetylcholinesterase activity. When pH-insensitive AuNCs were used as an internal standard, FITC/BSA-AuNCs offered a sensitive and reversible ratiometric sensing of a 0.1-pH unit change in the pH range 5.0-8.5. The pH-induced change in FITC fluorescence enabled FITC/BSA-AuNCs to detect an ammonia product-related enzyme system. This was exemplified with the determination of urea in plasma by urease-mediated hydrolysis of urea.
authors.affiliation: Department of Chemistry, National Sun Yat-sen University, Taiwan.
authors.forename: Chen-Yi
authors.initials: CY
authors.lastname: Ke
authors.affiliation: Department of Chemistry, National Sun Yat-sen University, Taiwan.
authors.forename: Yun-Tse
authors.initials: YT
authors.lastname: Wu
authors.affiliation: Department of Chemistry, National Sun Yat-sen University, Taiwan; School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan; Center for Stem Cell Research, Kaohsiung Medical University, Taiwan. Electronic address: tsengwl@mail.nsysu.edu.tw.
authors.forename: Wei-Lung
authors.initials: WL
authors.lastname: Tseng
mesh_terms: D001665:Binding Sites; D003460:Crystallization / Q000379:methods; D004798:Enzymes / Q000737:chemistry* / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D058726:Nanoconjugates / Q000737:chemistry / Q000648:ultrastructure; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D005456:Fluorescent Dyes; D058726:Nanoconjugates; D012710:Serum Albumin, Bovine; D006046:Gold; D006861:Hydrogen Peroxide; D016650:Fluorescein-5-isothiocyanate
keywords: Enzyme; Gold nanoclusters; Hydrogen peroxide; Ratiometric sensor; pH