A Thermostable Hydrogen Peroxide Sensor Based on "Wiring" of Soybean Peroxidase

Abstract

A thermostable soybean peroxidase-based biosensor was formed by cross-linking and electrically "wiring" the enzyme through a redox-conducting hydrogel to a glassy carbon electrode. At 65 °C the sensitivity of the sensor decayed only at a rate of only <2%/h. The sensor maintained, at 25 °C, the 1 A cm"1 2 M"1 sensitivity and the 0.1-200 µ dynamic range of the earlier "wired" horseradish peroxidase-based sensors.The life of a biosensor is often limited by that of its enzyme. YSI1 and others2 realize operational stability by designing sensors with an excess of enzyme and a membrane that reduces the flux of substrate. These devices maintain their performance specifications even after most of their enzyme has become inactive, as their residual enzyme suffices to react with the restricted substrate flux. Other stabilization efforts focused on slowing the inactivation of the enzyme. Assuming that inactivation resulted in irreversible changes in protein folding, researchers modified the protein surface or fixed its structure by cross-linking for better stability.3 4"5 Reaction of amino acid residues, known to contribute to instability, also extended the life of sensors in continuous operation.6 Superior thermostability resulted from covalent bonding of an enzyme in a phospholipid-modified surface, resembling the enzyme's natural environment in a membrane.7•8 Enhancing the thermostability of sensors by making them with enzymes isolated from thermophilic organisms has been considered,9 though these enzymes either were unavailable in the needed quantity or did not have the needed activity. Recently, a thermostable peroxidase isolated from soybean has become commercially available.10 **In tests by the manufacturer, this enzyme had a half-life of > 12 h at 80 °C.

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…Also, unlike HRP, SBP is active in the pH range [2][3][4][5][6]191 offering a greater range of potential biosensing applications [56]. The first SBP 192 biosensor was reported in 1995 by Vreeke and co-workers [57] as a thermostable 193 wired enzyme electrode using an osmium-based mediator, which aids electron transfer 194 from the active site to the electrode, modified by an epoxide. workers [58] also used a poly(4-vinylpyridine) polymer, complexing the pyridine 196 nitrogens to the osmium-based mediator, quaternised with 2-bromoethylamine.…”

Electrochemical Biosensing in Extreme Environment

“…However, these enzymes were unavailable in large amount. Recently, a thermostable peroxidase isolated from soybean has become commercially available, and Heller et al [9] constructed a thermostable hydrogen peroxide sensor based on wiring the soybean peroxidase to electrode through electron-conducting redox hydrogel. At 65 8C, the sensitivity of the sensor only decreased at a rate of 1.7%/h, while the wired HRP electrode decayed at a rate of 50%/h.…”

“…Three types of enzymes can be used for the construction of stable sensors. First, thermophilic enzymes such as soybean peroxidase and l-glutamate dehydrogenase have been purified from thermophilic microorganism [8,9], and the resulting biosensors exhibited enhanced stability at high temperature. Another type of enzyme is the artificial synzyme consisting of stable and synthetic polymers with functional moieties that mimic natural enzyme activity.…”

Acid-Stable Amperometric Soybean Peroxidase Biosensor Based on a Self-Gelatinizable Grafting Copolymer of Polyvinyl Alcohol and 4-Vinylpyridine

“…Two aspects can explain the high sensitivity of the SBP biosensor. On one hand, SBP is a more stable enzyme than HRP, accordingly the denaturalization tendency for SBP is lower than that for HRP [8,10]. On the other hand, the grafting copolymer contains a large amount of hydroxyl groups and hydrogen bonds, which is also favorable to maintaining the active conformation of SBP [12].…”