An amperometric bienzyme biosensor for rapid measurement of alanine aminotransferase in whole blood

A two-electrode configuration has been fabricated by depositing a gold film on a polyethylene terephthalate (PET) substrate. The integrated reference electrode was obtained by screen-printing commercial silver and silver chloride paste on the gold electrode. The working electrode was modified by drop-coating a redox polymer of poly(4-vinylpyrodine) complexed with osmium (2,2′-bipyrridine)2. The bienzymes (horseradish peroxidase and l-glutamate oxidase) were immobilized in the redox hydrogel matrices by the poly (ethylene glycol) diglycidyl ether (PEGDGE) crosslinker. The optimized biosensor was used for the amperometric measurement of l-glutamate in a stirred solution at 0 mV versus Ag|AgCl. A linear calibration graph was obtained in the concentration range of l-glutamate from 1 to 500 μM (linear regression coefficient = 0.99) with a steady-state response time of less than 10 s. The sensors were characterized by a sensitivity of 55.3 μA mM−1 cm−2 with an operational stability of more than 90% of its original activity over a period of 1 month. The alanine aminotransferase in real human serum and whole blood samples was analyzed subsequently with this sensor method and compared to the clinical method with satisfactory results.