A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase.
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.
Berberich
Jason A
JA
Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Yang
Lee Wei
LW
Madura
Jeff
J
Bahar
Ivet
I
Russell
Alan J
AJ
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
D004800:Enzymes, Immobilized; D012834:Silver; D011092:Polyethylene Glycols; D003404:Creatinine; D050879:Sarcosine Oxidase