Rapid determination of kappa-carrageenan using a biosensor from immobilized Pseudomonas carrageenovora cells

摘要

A potentiometric whole cell biosensor based on immobilized marine bacterium, Pseudomonas carrageenovora producing κ-carrageenase and glycosulfatase enzymes for specific and direct determination of κ-carrageenan, is described. The bacterial cells were immobilized on the self-plasticized hydrogen ion (H+)-selective acrylic membrane electrode surface to form a catalytic layer. Hydrogen ionophore I was incorporated in the poly(n-butyl acrylate) [poly(nBA)] as a pH ionophore. Catalytic decomposition of κ-carrageenan by the bienzymatic cascade reaction produced neoagarobiose, an inorganic sulfate ion and a proton. The latter was detectable by H+ ion transducer for indirect potentiometric quantification of κ-carrageenan concentration. The use of a disposable screen-printed Ag/AgCl electrode (SPE) provided no cleaning requirement and enabled κ-carrageenan detection to be carried out conveniently without cross contamination in a complex food sample. The SPE-based microbial biosensor response was found to be reproducible with high reproducibility and relative standard deviation (RSD) at 2.6% (n = 3). The whole cell biosensor demonstrated a broad dynamic linear response range to κ-carrageenan from 0.2-100 ppm in 20 mM phosphate buffer saline (PBS) at pH 7.5 with a detection limit at 0.05 ppm and a Nernstian sensitivity of 58.78±0.87 mV/decade (R2 = 0.995). The biosensor showed excellent selectivity towards κ-carrageenan compared to other types of carrageenans tested e.g. ι-carrageenan and λ-carrageenan. No pretreatment to the food sample was necessary when the developed whole cell biosensor was employed for direct assay of κ-carrageenan in dairy product.