Bienzymatic creatine biosensor based on reflectance measurement for real-time monitoring of fish freshness

Abstract

In view of the growing need for rapid and non-destructive method for monitoring of fish freshness to secure the safety standard of food, a reflectometric biosensor kit has been developed for in situ and real-time analysis of creatine as freshness marker. The optical bienzymatic creatine biosensor was fabricated by covalent grafting of both creatinase (CI) and urease (Ur) on the pH chromoionophore ETH 5295-doped poly(styrene-co-acrylic acid) (PSA) latex microspheres via EDC/NHS coupling chemistry. An optical fiber reflectance spectrophotometer was employed for reflectance measurement of the biorecognition phase for quantification of creatine levels, which can be used as the indicator of the extent of fish spoilage for direct evaluation of fish freshness. The dynamic linear response range of the reflectance creatine biosensor was obtained from 16 to 48 mM, which corresponds to the physiological range of creatine in fish body surface metabolite with a reproducibility of less than 2% relative standard deviation (RSD). The biosensor response time was about 7 min and did not show interference by some important biogenic amines (BAs), amino acids and nitrogen compounds normally co-exist with creatine in the biofluid of fish surface such as histamine, phenylalanine, tyrosine and taurine except sarcosine and uric acid.