Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible electrospun polymer nanofibers for use in chemiluminescent detection assays.
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 approximately 4 microm; mean diameter approximately 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II resulted in the highest enzyme loading (48.1 +/- 0.4 microg . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinking. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD. 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.
Mark
Sonny S
SS
Center for Biomedical Micro & Nanotechnology, Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. s.mark@axela.com
Stolper
Samuel I
SI
Baratti
Carla
C
Park
Jason Y
JY
Taku
Maria A
MA
Santiago-Avilés
Jorge J
JJ
Kricka
Larry J
LJ
D000218:Adamantane / Q000031:analogs & derivatives / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000737:chemistry*; D002209:Caprolactam / Q000031:analogs & derivatives / Q000737:chemistry; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D008867:Microtomy; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D013010:Sonication; D055672:Static Electricity
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011108:Polymers; C071872:3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; C009916:nylon 6; D002209:Caprolactam; D000469:Alkaline Phosphatase; D000218:Adamantane