Electrochemical biosensor based on polytyramine coated Prussian blue: Fabrication, electrochemical studies and potential analysis on glucose

Diabetes mellitus, one of the most significant threats to human health, is a metabolic disorder in case the body’s inefficiency to produce any or enough insulin causes high blood glucose levels. On the other hand, low blood glucose levels can cause hypoglycemia symptoms that are not enough glucose in blood. Therefore, the blood glucose level is very important in the early diagnosis and treatment of diabetes. Electrochemical enzyme biosensor, one of the interesting method, was widely used to measure glucose due to their benefits such as convenience, low cost, excellent selectivity and high sensitivity [1]. However, most of the enzyme immobilization approaches are by cross-linking resulting in the loss of some enzyme activity [2]. Therefore, a simple immobilization with high enzyme activity has been an interesting issue in the development of enzyme biosensors. This can be employed with gold nanoparticles (GNPs) since thiol and amino-containing enzymes can easily be adsorbed onto the GNPs [3]. To achieve this objective, different enzyme immobilization approaches were studied for amperometric glucose biosensors (Fig. 1). In this study, an amperometric biosensor for glucose was firstly developed by immobilizing glucose oxidase (GOx) on GNP that deposited on the layer of polytyramine (Ptyr)/Prussian blue (PB) modified on screen-printed carbon electrode (SPCE) using glutaraldehyde as traditional cross-linking agent. PB, known as an artificial peroxidase, has been widely employed in fabricating amperometric biosensors because of its excellent catalytic activity toward hydrogen peroxide (H2O2) reduction at a low potential [4]. In addition, Ptyr, a primary amino-containing compound, was used as supporting material for the enzyme immobilization due to its good biocompatibility, environmentally friendly, and good film-forming ability. Furthermore, the abundance of free amino groups on the nonconductive Ptyr thin film can covalently attach with GNPs [5]. Then, GNPs was further employed for the immobilization of GOx [6]. The use of GNPs not only enhances the performance of the electrochemical sensor but also achieves a reagentless and simple preparation. The electrochemical experiments of different modified electrodes, GOx/GNPs/Ptyr/PB/SPCE and GOx/Ptyr/PB/SPCE, were examined with cyclic voltammetry and amperometry. The produced H2O2 was measured at Ptyr/PB/SPCE and GNPs/Pty/PB/SPCE without immobilized GOx. In the addition of H2O2 concentrations, the increase in reduction current on Ptyr/PB/SPCE and GNPs/Ptyr/PB/SPCE, studied by cyclic voltammetry, were obtained due to the excellent catalytic activity toward H2O2 reduction of PB. Compared to Ptyr/PB/SPCE, the reduction current increase at GNPs/Ptyr/PB/SPCE confirming the excellent electrochemical characteristic behavior of GNPs that was modified on the electrode. In order to evaluate the performance of the biosensor, the electrodes were then immobilized with GOx that catalyzes the oxidation of glucose to form gluconolactone and H2O2 [7]. Amperometric determination of glucose in 0.10 M phosphate buffer solution (pH 7.0) containing 0.10 M KCl by the GOx/Ptyr/PB/SPCE and GOx/GNPs/Ptyr/PB/SPCE were carried out at an applied potential of −0.10 V (vs. Ag). The reduction currents of PB as a mediator were proportional to glucose concentration in the range of 0.010 ‒ 1.0 mM with glucose sensitivity of 1.182 ± 0.015 µA/mM at GOx/Ptyr/PB/SPCE and 1.4839 ± 0.0053 µA/mM at GOx/GNPs/Ptyr/PB/SPCE. All these electrochemical studies showed that GNPs/Ptyr/PB as sensing interface for the enzyme immobilization could alternatively be a promising material for the fabrication in enzyme biosensors.