Enzymatic Determination of Serum Urea on the Surface of Silicone-Rubber Pads

Abstract

An enzymatic, fluorometric method is described for determination of serum urea on silicone-rubber pads. In this method, the reagents are lyophilized on the surface of the pads, NADH on one side and a mixture of urease, glutamate dehydrogenase, and α-ketoglutarate on the other. The rate of disappearance of NADH fluorescence at 460 nm (excitation wavelength, 340 nm) is monitored and related to serum urea concentration. The calibration curve is linear to 250 mg of urea per liter. The method affords a rapid, simple, and inexpensive means for urea assay, the results of which correlate well with automatic diacetyl monoxime method (correlation coefficient, 0.998).

Analytical aspects of immobilized enzyme columns

“…In particular, it is readily possible to prepare several hundred active units of enzyme immobilized in a small (1 mL) column, thereby permitting equilibrium assays and circumventing many of the limitations of kinetic assays with soluble enzymes. Immobilized enzymes have been used in flow-through reactors of several configurations including: fixed-bed (18) and open tubular reactors (19), as well as on electrodes (20) and reagent pads (21).…”

Enzymic determination of nitrate: fluorometric detection after reduction with nitrate reductase

“…Therefore, this procedure can be a very useful method for monitoring of nitrate in contaminated drinking water, as well as in food and other fields. The main disadvantages generally associated with most fluorescence methods, i.e., (1) errors from fluorescing contaminants such as aromatic organic compounds in the water sample and (2) sample matrix effects generally caused by spectral and nonspectral interferences associated with the matrix, are not as important in solid-surface methods such as the one described herein. Any strongly fluorescing com-pounds present, which absorb or emit at the same wavelengths as NADH, would interfere.…”

“…Chlorella vulgaris was a kind gift from L. P. Solomonson, Department of Biochemistry, College of Medicine, University of South Florida. 1 Present address, Department of Chemical Engineering and Nuclear Engineering, Iowa State University, Ames, Iowa 50011.…”

“…, " nitrate reductase no3 + nadh2 +----------NOT + NAD+ + H20 (1) Because of its simplicity, sensitivity, and economy, a newly developed silicone-rubber pad fluorometric method (1) was used for all measurements.…”

Applications of immobilized enzymes in analytical chemistry

“…After reconstitution the analyte solution is placed on the pad and allowed to diffuse into the gel. An interesting example is found in the analysis of serum urea in which urease, glutamate dehydrogenase, and NADH are entrapped in the pad (21). Upon addition of serum, the hydrolysis of urea is coupled to the removal of NADH by the reactions: urease Urea + H+ + 2HaO ^=±; 2NH4+ + hco3-(D a-ketoglutarate + NH4+ glutamate + NADH NAD+ dehydrogenase + glutamate + H20 (2) Quantitation is accomplished by monitoring the change in fluorescence as a function of time.…”