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

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 (r ² = 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.