Amino-functionalized graphene quantum dots based ratiometric fluorescent nanosensor for ultrasensitive and highly selective recognition of horseradish peroxidase

Abstract

Herein, a ratiometric fluorescent nanosensor for ultrasensitive and highly selective recognition of horseradish peroxidase (HRP) was reported for the first time, by employing amino-functionalized graphene quantum dots (af-GQDs) as the reference fluorophore and 2,3-diaminophenazine (DAP) as the specific response signal. DAP was the oxidation product of o-phenylenediamine (OPD) that acted as the cosubstrate of HRP. Upon the addition of HRP, OPD could be catalytically oxidized to DAP, then the fluorescence intensity corresponding to DAP at 553nm increased dramatically with a simultaneous fluorescence quenching of af-GQDs at 440nm, resulting in a ratiometric fluorescent nanosensor toward HRP. This ratiometric fluorescent nanosensor exhibited a broad linear range and excellent sensitivity toward HRP detection. The fluorescence intensity ratio of DAP to af-GQDs linearly increased with the increasing of HRP concentration in the range of 2 fM–800 fM (0.02μUmL−1–8μUmL−1) with a detection limit down to 0.21 fM (2.1nUmL−1). The present ratiometric fluorescent nanosensor also showed excellent selectivity for HRP over some amino acids, nucleotides, and other heme-containing proteins. The enzymatic activity of HRP was further evaluated by quantitatively calculating the enzymatic velocity and Michaelis−Menten kinetic parameter.