Reagentless Amperometric Pyruvate Biosensor Based on a Prussian Blue- and Enzyme Nanoparticle-Modified Screen-Printed Carbon Electrode

Abstract

We report a facile strategy for developing reagentless amperometric pyruvate biosensors based on enzyme nanoparticles (EnNPs). The EnNPs were prepared using pyruvate oxidase crosslinked with graphene quantum dots. Before EnNP immobilization, screen-printed carbon electrodes (SPCEs) were modified with Prussian blue, a biocompatible coordination polymer. The biosensor system was optimized in terms of the working potential and pH value. At pH 7.0 in 50 mM phosphate-buffered solution, the biosensor showed optimal characteristics under an applied potential of -0.10 V versus an internal pseudo-Ag reference electrode. Using these optimized conditions, the biosensor performance was characterized via the chronoamperometric technique. The EnNP-immobilized SPCE exhibited a dynamic linear range from 10 to 100 μM for pyruvate solution, and a sensitivity of 40.8 μA mM^-1 cm^-2 was recorded. The observed detection limit of the biosensor was 0.91 μM (S/N = 3) and it showed strong anti-inference capability under the optimized working potential. Furthermore, the practical applicability of the proposed biosensor was studied in fish serum samples.

Figure 1

FE-SEM images of (a) bare SPCE, (b) PB/SPCE, and (c) PoxBNP/PB/SPCE. Scale bar: 3 μm.

Figure 2

(a) Cyclic voltammograms of the bare SPCE, GQD/SPCE, PB/SPCE, and GQD/PB/SPCE in N₂-saturated 50 mM PBS (pH 7.0) without (dash line) and with (solid line) 0.5 mM H₂O₂ at a scan rate of 10 mV s^–1 and (b) corresponding plot of i_pc values measured at −0.10 V for the electrodes with different modifications.

Figure 3

(a) Current change under different applied potentials for the bare SPCE, PB/SPCE, and GQD/PB/SPCE in 50 mM PBS (pH 7.0) containing 0.1 mM H₂O₂. (b) Current change under different pH values for PoxBNP/PB/SPCE in 50 mM PBS containing 0.25 mM Py at an applied potential of −0.1 V.

Figure 4

Amperometric responses of (a) PoxB/SPCE, PoxBNP/SPCE, PoxB/PB/SPCE, and PoxBNP/PB/SPCE; (b) GQD/PB/SPCE and PoxBNP/PB/SPCE in 50 mM PBS (pH 7.0) without (dash line) and with (solid line) 0.5 mM Py at an applied potential of −0.1 V.

Figure 5

(a) Representative amperometric responses of the PoxBNP/PB/SPCE with successive addition of Py to 50 mM PBS (pH 7.0) at an applied potential of −0.1 V. (b) Calibration plot obtained from (a).

Figure 6

Amperometric response of the PoxBNP/PB/SPCE in 50 mM PBS (pH 7.0) at an applied potential of −0.1 V, with successive additions of 0.5 mM Py, 1.0 mM glutamate, 1.0 mM acetoacetate, 1.0 mM myo-inositol, 1.0 mM glucose, and 0.5 mM Py.