(375e) Enzymatic Electrochemical Sensing for Fish Freshness Using Macro-Porous NiO Electrodes

The freshness of fish meat is the most important criterion in the pre-consumption quality control process. Complex microbiological processes lead to the loss of freshness and subsequent spoilage. Six hours after death, autolytic decomposition of adenosine triphosphate (ATP, the energy-storing molecule found in cells) begins to produce xanthine (XA) as one of the primary metabolites. XA concentration increases with storage time, and thus it can be used as an indicator of fish freshness.

Current XA determination methods, which use chromatography, colorimetry, spectrophotometry, or mass spectrometry, are time-consuming, expensive, and require skilled operators. Electrochemical sensor platforms, on the other hand, are attractive because of their ease of use, low cost, small sample volume requirements, and quick analysis. In recent years, enzyme-based electrochemical biosensors, employing xanthine oxidase (XO) for selective recognition and catalysis of XA, have become promising for XA determination in fish and meat samples because of their low detection limits, high selectivity, and sensitivity.

The working electrode material has a significant impact on the analytical performance of electrochemical biosensors, and the integration of nanomaterials in the working electrode has previously improved sensor performance. Although both carbon-based and metal/metal oxide nanostructures with favorable electrical characteristics and biocompatibility have been reported for enzymatic electrochemical biosensor fabrication, macroporous NiO electrodes are rarely employed for enzymatic detection of XA.

Macroporous NiO electrodes are ideal for electrochemical biosensor fabrication owing to their large surface area for enzyme immobilization, high isoelectric point, biocompatibility, chemical stability and fast electron transfer features. Furthermore, NiO possesses its own redox pair (Ni³⁺/Ni²⁺), allowing for reagentless biosensing. Here, we describe macroporous NiO electrodes fabricated by glancing angle deposition (GLAD). GLAD is a physical vapor deposition technique capable of producing meso- and macro porous thin films with extremely high surface area and internal porosity. The large surface area and porosity of the GLAD films should allow immobilization of large quantities of the XO enzyme, which, when combined with the excellent electrical properties of the GLAD electrode, is expected to allow for the amperometric detection of XA. In this work, XO is immobilized on a NiO GLAD electrode, and the resulting sensor is used to determine the concentration of XA in a fish sample with excellent sensitivity, detection limit, and response time.

Sensor fabrication and performance

Nanocolumnar and mesoporous NiO films (thickness ~500 nm) were deposited on ITO using GLAD. XO was physisorbed onto electrochemically activated GLAD NiO electrode by incubating in XO-loaded PBS buffer, pH 7.4 at room temperature. The modification of the NiO GLAD film with XO was characterized by electrochemistry and X-ray photoelectron spectroscopy. XO catalyzes the oxidation of XA to uric acid and hydrogen peroxide (H₂O₂), and therefore, using an optimized measurement condition, the current was monitored by applying +0.5 V vs Ag/AgCl to the electrode. This current was found to be proportional to the concentration of XA. The amperometric sensor rendered a dynamic range of 0.1 µM to 650 µM, with a limit of detection of 37 nM, good reproducibility (relative standard deviation of ~ 4%, n=18), rapid response time (~7 s), and a high sensitivity (1.1 µA·µM^-1·cm^-2 in the low concentration range from 0.1-5 µM, and 0.3 µA·µM^-1·cm^-2 in the higher concentration range from 5-650 µM) compared to the flat NiO-based enzymatic sensor (0.03 µA·µM^-1·cm^-2). The sensor showed little interference from common fish sample matrices (such as glucose, uric acid, hypoxanthine) and fish preservative (sodium benzoate), and was used to quantify XA in real fish samples. The sensor could also be stored in buffer at 4^oC for over a week without losing its performance.

Overall, the developed enzymatic electrochemical biosensor exhibited good performance to monitor fish spoilage. We believe this study will contribute to a better understanding of macro-porous NiO electrodes, which will aid in the development of electrochemical biosensors for other compounds.