Polyelectrolyte-Assisted Immobilization of Active Enzymes on Glass Beads

Abstract

Using alternating layers of charged polymers, we have constructed reactive thin films by incorporating enzymes, specifically alkaline phosphatase (AP) and glucose oxidase (GOD), into multilayers of a polycation, branched polyethylenimine (PEI), and a polyanion, poly(styrenesulfonate) (PSS), supported on a glass substrate. Experiments using a quartz crystal microbalance (QCM) demonstrated that the films grew sequentially on a solid support, while X-ray photoelectron spectroscopy (XPS) further confirmed the growth and deposition of successive enzyme layers. For both enzymes, the reactive films demonstrated increased activity with the successive number of deposited enzyme layers. In hybrid films, consisting of alternating layers of AP and GOD, both enzymes retained activities similar to those of their corresponding films of either enzyme alone. The effect of elevated temperature was also investigated for these reactive films. Increased thermal stability was found to be associated with the increase in the number of deposited enzyme layers.

Performance of Poly(styrenesulfonate)‐Coated Thin Mercury Film Electrodes in the Determination of Lead and Copper in Estuarine Water Samples of High Salinity

“…Poly(estersulfonic acid) coatings were basically used for the fabrication of glucose sensors [18] and more recently mercury thin film electrodes coated with the same polymer were used for characterization and application in batch injection analysis stripping voltammetry of heavy metal ions [19]. Poly(styrenesulfonate) (PSS) membrane incorporating immobilized enzymes was successfully used in amperometric biosensors [12,13,16,20,21]. Thin films of PSS on glassy carbon electrodes were also used in studies of the [Os III (bipy)]…”

Bioconjugation of Alkaline Phosphatase to Mechanically Processed, Aqueous Suspendible Electrospun Polymer Nanofibers for Use in Chemiluminescent Detection Assays

“…Santos et al, for example, successfully employed the LBL technique to produce glass beads (30-50 mm diameter) coated with 2, 5, 8 and 11 layers of ALP enzyme. [20] As successive layers of enzymes were deposited, there was a monotonic increase in the amount of enzyme adsorbed per milligram of glass beads and, consequently, a correspondingly higher amount of catalytic activity detected per bead. And finally, to potentially improve the enzyme loading efficiency on the nanofibers through covalent-based immobilization methods, the use of alternative types of crosslinking chemistries may be considered.…”

“…As such, it is usually desirable to test a range of alternative bioconjugation strategies whenever a new enzyme/immobilization support system is explored. Of particular relevance for this study, alkaline phosphatase (ALP) has been previously immobilized by a variety of methods to inorganic (silica) beads [20,21] /fibers, [22] polymeric microspheres [23] and carbon nanotubes. [14,15,24] These studies successfully demonstrated the assembly of large numbers of functionally active ALP onto a range of different types of supports; however, these investigations did not examine the possibility of using ALP immobilized to micro-/nanostructures to catalyze the decomposition of chemiluminescent substrates.…”

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays

“…bilized ALP molecules by simply adsorbing a larger number of alternating polycationic Sapphire II TM /ALP bilayers during the LBL assembly process. Santos et al, for example, successfully employed the LBL technique to produce glass beads (30-50 m diameter) coated with 2, 5, 8 and 11 layers of ALP enzyme [15]. As successive layers of enzymes were deposited, there was a monotonic increase in the amount of enzyme adsorbed per milligram of glass beads and, consequently, a correspondingly higher amount of catalytic activity detected per bead.…”

“…Alkaline phosphatase (ALP) has been previously immobilized by a variety of methods to inorganic (silica) beads [15,16]/fibers [17], polymeric microspheres [18] and carbon nanotubes [9,10,19]. These studies successfully demonstrated the assembly of large numbers of functionally active ALP onto a range of different types of supports; however, these investigations did not examine the possibility of using ALP immobilized to micro-/nanostructures to catalyze the decomposition of chemiluminescent substrates.…”