Photometric and Fluorimetric Determination of Creatine Kinase Activity by Using Co-Immobilized Auxiliary Enzymes and an Open/Closed Flow Injection Manifold

Abstract

An open-closed configuration for the determination of creatine kinase activity based on fixed-time and reaction-rate measurements on a multipeak recording obtained by using a single conventional photometric or fluorimetric detector is proposed. The complexity of the biochemical reaction (three enzyme-catalysed steps) was partially overcome by using the two auxiliary enzymes that catalyse the two-step indicator reaction co-immobilized on controlled-pore glass. Both measurement methods provided excellent results (linear calibration ranges 0.01–0.80 U/L and 0.01–2.00 U/L, and r.s.d. smaller than 2%) and were checked by application to the determination of the analyte activity in serum samples. The results obtained agreed well with those provided by the standard method, and recoveries were between. 96 and 103% in all instances.

Key Words:

• FIA
• open-closed system
• enzyme activity
• immobilization
• fluorimetry
• photometry