A rapid and sensitive fluorometric flow injection assay for subtilisin-type enzymes utilising sol-gel particles directly coated with gelatin–Texas Red substrate

Abstract

Sol-gels are becoming widely used as matrices in a variety of analytical systems. A simple method of immobilising fluorophore-labelled protein substrate has been developed which, when packed in the form of a bioreactor has resulted in a rapid and sensitive assay for subtilisin-type enzymes. The immobilisation process is based on adsorption of a Texas Red–gelatin conjugate onto the surface of the sol-gel particles via strong ionic interactions. This direct coating circumvents the need to coat via covalent binding between the protein and activated surface groups on the sol-gel surface. Exposure of the bioreactor, located in a FIA system, to subtilisin-type enzymes resulted in fluorescent peaks that were detected downstream after 4–5 min. Linear responses to the enzyme savinase over the range 20–200 µg l⁻¹ buffer (r² = 0.9985, n = 4) were achieved with a limit of detection of 17.0 µg l⁻¹. The system showed excellent specificity for the subtilisin-type enzymes savinase and purafect, with no response to the non-subtilisin type enzymes papain, cellulase and lipase.