Gold nanoclusters (AuNCs) have been covalently linked to a FAD-dependent enzyme, choline oxidase (AuNCs@ChOx), with the objective of using the AuNCs fluorescence at 700 nm for monitoring the enzymatic reaction. The linking methodology has been optimized to obtain oriented binding and as a result the structure of the enzyme changes leading to an enhancement of the intrinsic FAD fluorescence of the enzyme (at 525 nm). During the enzymatic reaction with choline, the AuNCs@ChOx fluorescence intensity at both wavelengths (700 nm and 525 nm) changes proportionally to the choline concentration and can be simultaneously monitored. The mechanism of the analytical signal has been studied and a combination of FRET and O2 quenching was found to be responsible for the changes. A mathematical equation is proposed for modelling the signal. The combination of AuNCs@ChOx with acetylcholinesterase permitted the determination of acetylcholine in a concentration range from 1·10−6 to 1·10-5 M and RSD = 4%. This new approach can potentially be applied to any other flavo-enzyme in order to both increase their FAD fluorescence properties and to move the fluorescence measurement to the NIR region. The biosensor is reversible and avoids the use of conventional fluorophores, which are prone to chemical degradation and present worse biocompatibility.

This work was supported by the MINECO of Spain (project CTQ2016-76846R) and research funding from DGA (Fondo Social Europeo) (E25_17R).

Peer reviewed