Microfabricated amperometric creatine and creatinine biosensors

Miniaturized enzyme electrodes for determination of creatine and creatinine in serum are described. The creatine sensor is based on a bienzyme sequence, involving creatine amidinohydrolase (CI) and sarcosine oxidase (SO). For the creatinine sensor a third enzyme, creatinine amidohydrolase (CA), is added. The signals of both sensors are based on the oxidation of the hydrogen peroxide formed in the enzymatic layer to yield currents proportional to the concentration of creatine or creatinine in the sample solution. For the optimization of enzyme loadings several macro-size electrodes were tested. Crosslinking with glutaraldehyde was selected as the immobilization method for the enzymatic system. The substrate for the miniaturized sensors is a polyimide foil, on which film structures are built using well established microelectronic techniques. Biosensors based on this planar, thin and thick film technology, have shown extended linear range (up to 2 mM), good sensitivity and operational stability (at least 3 months) when tested in buffer solutions. A cellulose acetate inner membrane in combination with a polyurethane outer membrane significantly reduces in vitro response of the biosensors to common interferences such as ascorbic acid, uric acid and acetaminophen. The creatine and creatinine microfabricated sensors were tested in control human serum samples, where the detection limit was 20 μM and 30 μM, respectively. The experimental values are in agreement with the assigned values for creatinine in the control samples.