Fluorescein-5-isothiocyanate-conjugated protein-directed synthesis of gold nanoclusters for fluorescent ratiometric sensing of an enzyme–substrate system

• Published in: Biosensors & bioelectronics 2015-07, Vol.69, p.46-53
• Main Authors: Ke, Chen-Yi, Wu, Yun-Tse, Tseng, Wei-Lung
• Format: Article
• Language: English
• Subjects: Binding Sites; Crystallization - methods; Detection; Enzyme; Enzymes; Enzymes - chemistry; Enzymes - ultrastructure; Fluorescein-5-isothiocyanate - chemistry; Fluorescence; Fluorescent Dyes - chemistry; Glucose; Gold - chemistry; Gold nanoclusters; Hydrogen peroxide; Hydrogen Peroxide - analysis; Hydrogen Peroxide - chemistry; Hydrogen-Ion Concentration; Inhibition; Metal Nanoparticles - chemistry; Metal Nanoparticles - ultrastructure; Nanoconjugates - chemistry; Nanoconjugates - ultrastructure; Nanostructure; Protein Binding; Protein Interaction Mapping - methods; Ratiometric sensor; Serum Albumin, Bovine - chemistry; Spectrometry, Fluorescence - methods; Substrate Specificity; Synthesis
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2015.02.002

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. •A facile method was introduced to synthesize dual emission of FITC/BSA-AuNCs.•FITC/BSA-AuNCs were used for ratiometric sensing of H2O2-related enzyme activity.•FITC/BSA-AuNCs were used for ratiometric sensing of environmental pH change•FITC/BSA-AuNCs could detect a 0.1-pH unit change in the pH range 5.0–8.5.