Stabilization of Glucose Oxidase and Alcohol Oxidase Based Biosensors By Immobilization in Electrochemically Generated Poly-o-Phenylenediamine Under High Hydrostatic Pressure
Authors: Yang Daoyuan , Olstad Hanna , Jenkins Daniel McKewn , Reyes De Corcuera Jose I 
Published: February 2020

Electrochemical biosensors have applications in medical diagnostic, agriculture, and food. In the food industry, glucose oxidase (GOX) and alcohol oxidase (AOX) based biosensors can be valuable for real-time detection and quality control of glucose and ethanol. The global market for electrochemical biosensors are expected to reach $23.7 billion by 2022 and thousands of research papers have been published annually since 2011. However, compared to the number of publications, there are very few commercial electrochemical enzyme-based biosensors currently available on market. Glucose and lactic acid biosensors are the most common commercial devices. High price and low stability of other enzymes are arguably the two main limiting factors. Therefore, it is critical to improve the biosensor stability. Enzyme covalent immobilization, addition of solutes like sugars and polyols, and crosslinking have been used for stabilization of enzyme biosensors. High hydrostatic pressure (HHP) has also been used to stabilize enzymes but not for biosensor fabrication because upon depressurization, enzymes return to their native, less stable state. Here we report the fabrication of stabilized of GOX and AOX based biosensors using entrapment under HHP. An electrochemical cell was fabricated to fit for the HHP reactor with a custom-made electrical feed-through connectors rated to up to 700 MPa (model U111, Warsaw, Poland). Two platinum wires (diameter 0.8128 mm) were inserted into a piece of Teflon rod to be the working and counter electrode. One Ag|AgCl electrode pellet was held in a heat shrinking tube (filled with 3 M KCl) with a porous glass frit to be the reference electrode. The platinum electrodes were polished and platinized before using. GOX or AOX was entrapped by electrochemical polymerization in 5 mM o-phenylenediamine at 650 mV vs. Ag|AgCl (3 M KCl) at 0.1 to 420 MPa or 0.1 to 240 MPa, respectively. The GOX biosensors were heated at 70 °C for 180 min and amperometric response was measured at 700 mV vs. Ag|AgCl (3 M KCl) every 20 min in 20 mM glucose solution. The AOX biosensors were heated at 50 °C for 10 min and amperometric response was measured at 700 mV vs. Ag|AgCl (3 M KCl) every 2 min in 100 mM ethanol solution. The amperometric response was measured using a commercial potentiostat (Reference 600, Gamry, Warminster, PA) or a low-cost portable wireless (Bluetooth) potentiostat developed in house (ABE-stat, University of Hawaii). After 180 min, the measured current decreased to 18.8% for GOX biosensors immobilized at 0.1 MPa, while to 47.3% for those immobilized at 420 MPa. After 10 min, the measured current decreased to 23.3% for AOX biosensors immobilized at 0.1 MPa, while to 34.8% for those immobilized at 240 MPa. Electrochemical impedance spectroscopy results showed no significant difference of the charge transfer resistance between GOX biosensors immobilized at 0.1 MPa and 180 MPa. To our knowledge, this is the first report of using HHP to improve the stability of enzyme-based biosensors. In the range of 0.1 to 420 MPa or 0.1 to 240 MPa, GOX and AOX biosensor stability increased with increasing pressure, respectively. The combination of HHP with cross-linking or chemical modification to further increase the stability of enzyme based biosensors is presented as well.