title: A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase.
abstract: The determination of creatinine levels in biological fluids is an increasingly important clinical requirement. Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to amperometrically measurable hydrogen peroxide. The development of the amperometric creatinine biosensor has been slow due the complexity of the three-enzyme system. This paper, the first of three, discusses the chemical modification of sarcosine oxidase and the immobilization and stabilization of this enzyme using polyurethane prepolymers. Sarcosine oxidase was completely inactivated after modification using poly(ethylene glycol) activated with isocyanate. The addition of a competitive inhibitor during enzyme modification was effective in protecting the enzyme from inactivation. Computational analysis of the structure of sarcosine oxidase suggests that there is a lysine in the active site that may be hyper-reactive. The enzyme was irreversibly immobilized using polyurethane prepolymers and retained significant activity. The enzyme's half-life at 37 degrees C increased from seven days to more than 50 days after immobilization.
authors.affiliation: Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Berberich
authors.forename: Lee Wei
authors.initials: LW
authors.lastname: Yang
authors.forename: Jeff
authors.initials: J
authors.lastname: Madura
authors.forename: Ivet
authors.initials: I
authors.lastname: Bahar
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Russell
mesh_terms: D015374:Biosensing Techniques; D003404:Creatinine / Q000032:analysis*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*; D050879:Sarcosine Oxidase / Q000737:chemistry*; D012834:Silver / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004800:Enzymes, Immobilized; D012834:Silver; D011092:Polyethylene Glycols; D003404:Creatinine; D050879:Sarcosine Oxidase