Abstract
The global food waste problem, especially aquatic product spoilage, stimulates the accurate freshness analysis of food products. However, it still remains a great challenge to realize in-field determination of fish freshness at the time of use. In the present study, a colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine (Hx), which is an important intermediate of adenosine triphosphate decomposition during fish storage. We demonstrate that xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity. It can oxidize different peroxidase substrates, including 3,3′,5,5′-tetramethylbenzidine, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine in the presence of H2O2, producing visible color reactions. Further experiments indicate that XOD-ASS displayed effective peroxidase activity and could be used for H2O2 detection. Based on this, a one-step Hx detection method was established using only XOD-ASS as the catalyst. The method displays a good linear relationship in the range from 20 to 100 μM with a detection limit of 6.93 μM. Additionally, we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage times. The recovery values range from 97.44 to 102.56%. It is exciting to note that, compared with other methods, our proposed method provides a robust advantage on the economic reaction system, ease of preparation, short time consumption, and moderate reaction temperature. We believe that this method shows good application prospects for on-site fish freshness determination.
Highlights
Nowadays, there are increasing requirements and regulations in the fields of biotechnology control, environmental protection, and food/water quality certification (Flachsbarth et al, 2015)
We find that xanthine oxidase (XOD) grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity; it can catalyze the oxidization of Hx or xanthine to produce H2O2, but catalyze the oxidization of 3,3,5,5 tetramethylbenzidine (TMB) by H2O2 to produce oxidized TMB
No color signal was observed for the reactions without xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) or H2O2, indicating XOD-ASS and H2O2 were both necessary for the oxidation of TMB
Summary
There are increasing requirements and regulations in the fields of biotechnology control, environmental protection, and food/water quality certification (Flachsbarth et al, 2015). A series of colorimetric or fluorescent biosensors based on the production of H2O2 by XOD and peroxidase-like catalytic activity of nanoenzymes, including platinum nanoparticles (Chen et al, 2020), selenium-doped graphite carbon nitride (Qiao et al, 2015), and amino-functionalized metal organic framework (Hu et al, 2018) are established, contributing to the rapid freshness evaluation of aquatic products In these reactions, a two-step catalytic reaction is adopted: First, XOD catalyzes the reaction of Hx or xanthine with oxygen, yielding H2O2; second, H2O2 participates in the peroxide reaction catalyzed by a peroxidase or peroxidase mimic enzyme. We demonstrate the utility of our method in fish samples and realize on-site, quantitative detection of Hx, providing a cheap, easy fish-freshness evaluation method
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