A novel strategy for the detection of pyruvate in fermentation processes based on well-distributed enzyme-inorganic hybrid nanoflowers on thiol graphene modified gold electrodes

We report a facile strategy for the detection of pyruvate in fermentation progress based on a non-enzymatic decarboxylation reaction of pyruvate with hydrogen peroxide (H 2 O 2 ), coupled with a horseradish peroxidase (HRP)-incorporating nanomaterial as an enzyme element to replace conventional pyruvate oxidase (POX). HRP-Cu 3 (PO 4 ) 2 •3H 2 O nanoflowers (HRP-CuP NFs) and thiol graphene (tG) were immobilized onto an Au disk electrode via layer-by-layer assembly to construct an improved amperometric H 2 O 2 biosensor. The as-prepared HRP-CuP NF/tG/Au electrode exhibited a high electrocatalytic performance toward H 2 O 2 , showing a sensitive response of 67.6 μA mM -1 cm -2 , as well as a linear range from 0.1-8.2 mM with a detection limit (LOD) of 0.06 mM (S/N=3). Furthermore, the resulting biosensor also demonstrated a linear range for pyruvate detection from 1.0 to 8.0 mM with LOD of 0.3 mM (S/N=3), under a working potential of -0.3 V. Finally, the biosensor was successfully used for the determination of pyruvate in real microbial fermentation processes without pretreatment, showing an excellent correlation coefficient compared to the standard HPLC assay with RSD values of 3.03-6.45%. This work provides a new approach for the design and preparation of portable sensors for indirect pyruvate detection with high performance, low cost, and easy operation.