Electrochemical determination of total alkaline phosphatase in human blood with a micropatterned ITO film

A voltammetric determination of alkaline phosphatase (ALP), a well-known enzyme in human blood, was investigated to survey the feasibility for a health diagnosis sensor. Simple photolithography with an office printer and transparency film was employed to construct an electrochemical sensor with an Indium tin oxide (ITO) film on glass. ITO films can be easily patterned with a resolution of 50 μm, and an integrated sensor consisting of working, counter and reference electrodes on a single ITO plate was constructed by this method. We used p-nitrophenyl phosphate (PNPP) as the substrate for ALP. PNPP was hydrolyzed enzymatically, and the product p-nitrophenol was detected by cyclic voltammetry and square wave voltammetry at an oxidation potential of +1.1 V (versus a fabricated Ag∣AgCl reference electrode) on a bare ITO electrode. According to this method, ALP can be detected in various media including fetal bovine serum, human serum and untreated human blood. The linear dynamic range of ALP was 5-180 units per liter (U/L). This method showed a good enough performance for applications and was sufficiently reproducible. Results were further examined via comparison with medical standard colorimetric analysis.