Alkaline phosphatase 4-aminophenyl phosphate substrate

E.J. Moore, M. Pravda, M.P Kreuzer, and G.G. Guilbault, Comparative study of 4-aminophenyl phosphate and ascorbic acid 2-phosphate, as substrates for alkaline phosphatase based amperometric immunosensor. Anal. Lett. 36, 303-315 (2003). 

Alkaline phosphatase is one of the most suitable enzymes for electrochemical immunoassays owing to its high turnover number and broad substrate specificity. Different substrates have been used, but 4-aminophenyl phosphate is most suitable, since the reaction product, 4-aminophenol is easily oxidized without fouling of the electrode surface. Thyroxine-binding globulin, cortisol, and prostatic acid phosphatase have been detected by using alkaline phosphatase. 

The amplification of the amperometric response of redox species has been studied for p-aminophenol The sensitive detection of this compound is of particular interest since it is the basis for the measurement of enzymes such as alkaline phosphatase and (J-galactosidase, which are frequently used in enzyme immunoassays The electrode active species (p-aminophenol) is liberated in the enzyme catalysed hydrolysis of its aminophenylated substrate which itself exhibit an irreversible electrochemical behavior The utility of p-aminophenyl phosphate in enzyme immunoassays in combination with IDA electrodes has been demonstrated in [6] The unfavorable pH-optimum of alkaline phosphatase is the drawback of this system Because of its neutral pH-optimum p-galactosidase appeared to be more advantageous An illustration of the proposed electrochemical enzyme immunoassay is given in Fig 2... 

The enzyme labels are typically horse radish peroxidase (HRP, electrochemical detection shown in the scheme) and alkaline phosphatase (ALP). Lor ALP, the selection of substrates is limited and p-aminophenyl phosphate (PAPP) is mostly used as the resulting aminophenol becomes easily and reversibly oxidised at the electrode. A wide choice of substrates (DH2) generating electroactive products includes hydroquinone, tetramethylbenzidine, aminosalicylic acid and iodide in the case of HRP ... 

When a carbon fiber microelectrode modified with alkaline phosphatase was immersed in a solution containing 4-aminophenyl phosphate, a steady-state current (at an applied potential of +0.30V this potential was employed in order to ensure mass transport control) due to the oxidation of 4-aminophenol was reached within 3-4 sec. in cating a rapid response. Moreover, the magnitude of the steady state current was dependent on the solution concentration of 4-aminophenyl phosphate. The response was linear at the lower concentrations (Figure 5) and (asymptotically) reached a saturation limit (as expected) for substrate concentrations above 20mM. From the linear portion of the calibration curve, a sensitivity of 1.4x10 nA M and a limit of detection of 5x10"were determined. These values are, in fact, superior to those that we had previously obtained at conventionally sized electrodes. 

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