Abstract
Application of enzymes as an analytical tools has recently been an exponential increase particularly in the field of clinical chemistry. Among them, of special interest and importance is the use of immobilized enzymes, which offer several advantages over conventional methods with soluble enzymes: thermal stability, resistance to decomposition and ease to assemble in an automatic instrument.
On the other hand, many clinically important compound in blood are oxidized by an individual oxidase with concomitant formation of hydrogen peroxide. For the detection of enzymically generated hydrogen peroxide, luminol chemiluminescence method is the most favorable because of its high sensitivity, wide linear dynamic range and simple instrumentation. Furthermore the reagents are relatively inexpensive and stable, therefore, this enable us to couple the enzymic process and the chemiluminescence detection process through the continuous flow system. This is very important because the use of an immobilized enzyme column is probably only one possible way to perform the enzyme catalyzed process in the continuous flow system.
As the first step toward the development of an automatic multichannel enzymic analyzer, we have assembled an apparatus which enable us to analyze glucose and uric acid in blood sample with high sensitivity. The appratus consists of immobilized oxidase column and chemiluminescence detector. All reagents, luminol, ferricyanide and buffer solutions, are supplied continuously by a peristaltic pump, and blood samples are introduced into enzyme column from an autosampler or injected by microsyringe. In the enzyme column, glucose oxidase or uricase, which are immobilized onto long chain alkyl amine glass by the cyanogen bromide method, are packed.
Enzymically formed hydrogen peroxide are mixed with chemiluminescence reagents in a swirling cell mounted directly on the surface of window of photomultiplier.
This apparatus has been found to be successfully applied for the quantitative determination of glucose and uric acid in blood samples in a range between 10-8 and 10-4 M.
By slight modification of the apparatus NAD (P) H was also determined using methylene blue as an electron carrier between NAD (P) H and oxygen. This system responded to the concentration of NAD (P) H down to 10-8 M.
