Aqueous suspendible polymer nanostructures were prepared by simple microtome processing of electrospun nylon 6 nanofibers and were used to immobilize calf intestinal alkaline phosphatase (ALP) by either covalent or noncovalent bioconjugation chemistries. It was found that noncovalent immobilization of ALP to the mechanically cut nanofibers (mean length approximate to 4 mu m; mean diameter approximate to 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II (TM) resulted in the highest enzyme loading (48.1 +/- 0.4 mu g . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinkine. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD (TM). The results indicate that the kinetic parameters, K-m and V-max for the catalytic activity of the nanostructure-bound ALP enzyme were influenced by the particular types of immobilization methods employed. In terms of the overall catalytic performance of the various immobilized ALP systems, a single-stacked LBL assembly approach resulted in the highest level of enzymatic activity per unit mass of nanofiber support. To the best of our knowledge, this study represents the first report examining the preparation of mechanically shortened, aqueous dispersed electrospun polymer nanofibers for potential application as enzyme scaffolds in chemiluminescent-based assay systems.