Integrating amino acid oxidase with photoresponsive probe: A fast quantitative readout platform of amino acid enantiomers

Abstract

Low-cost, high-throughput, broadly useful photoresponsive enantiomeric excess (ee) sensing of amino acids remains challenging to date. Herein, based on the selective oxidation reaction of amino acid oxidase (AAO) to amino acid enantiomers (D/L-AA) and the oxidation reaction of substrate (H2O2) with aromatic boronic ester, we put forward a photoresponsive strategy for the determination of D/L-AA at a certain concentration. In this scheme, the substrate H2O2 produced by the enzyme-catalyzed reaction was determined by sensitive fluorescent and colorimetric response of ethyl-3-(3-(benzothiazol-2-yl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)-2-cyanoacrylate (HBT-PB) to reflect the enantiomeric content at a certain concentration. The photoresponsive probe HBT-PB was readily available and inexpensive with sensitive long-wavelength red fluorescence and colorimetric light response to H2O2, the detection limit (LOD) was estimated as 2.91 μM. The operation of the sensing method was simple and data collection and processing are straightforward. The practicability of the scheme was favorably confirmed by accurate and scientific analysis of methionine and Dopa samples. As a result, the scheme was not only suitable for high-throughput screening but also adaptable to low-cost and sensitive RGB colorimetric analysis platform (LOD of methionine and Dopa was calculated as 9.23 μM and 8.34 μM respectively) with modern plate readers, and possessed extremely high enantioselectivity and wide applicability which benefited from the specificity and efficiency of enzyme catalytic reaction.