Fluorometric enzymatic determination of serum creatinine on the surface of silicone-rubber pads

An enzymatic semi-solid surface fluorometric method is described for the determination of serum creatinine on silicone-rubber pads. In this method, the glutamate dehydrogenase, alpha-ketoglutarate, ADP and NADH are mixed, then 30 microliters of diluted serum is added. After the free ammonium ion in serum is consumed, creatininase is added to initiate the assay. The rate of disappearance of NADH fluorescence at 460 nm (excitation wavelength 340 nm) is monitored and is proportional to the serum creatinine concentration. The whole assay uses only 0.41 I.U. creatininase, and takes less than 5 min. The calibration curve is linear up to 82 mg creatinine per liter. The proposed method offers a rapid, simple and inexpensive means for creatinine assay. The results obtained correlate well with the modified Jaffe method applied on Technicon SMA 12/60, with a correlation coefficient of 0.998. The recovery averages 99.3%.