A sequential injection capillary immunoassay system (SI-CI) was developed to automate the multi-step competitive bioassay procedures for hyaluronan (HA), a potential biomarker for liver and cancer diseases. Biotinylated HA-binding proteins (b-HABPs), anti-biotin conjugated with horseradish peroxidase (anti-b-HRP) and substrate 3,3′,5,5′ tetramethylbenzidene (TMB) were employed. An ordinary glass capillary was utilized as a bio-reactor where competitive assay took place. Standard HA was immobilized onto an unmodified glass capillary through simple physical adsorption. This glass capillary is much lower cost compared to a fused-silica capillary, and is well suited for use as part of the tubing of a flow system. Using the proposed system, the average amount of HA in human serums from patients with liver disease and healthy subjects could be differentiated with comparable efficiency as the conventional micro-plate assay (as compared by t-test), with an improved assay time per sample (20 min vs. 5 h), and with approximately 10 times less reagent consumption (10 − 80 μl vs. 100 − 350 μl).
2010 by The Japan Society for Analytical Chemistry