Development of an electrochemical immunosensor for alanine aminotransferase.

Alanine aminotransferase (ALT) has been regarded as one of the most sensitive indicators of hepatocellular damage. While ALT is widely used in the practice of medicine, few attempts have been made to develop biosensors applicable to the on-site diagnosis of liver diseases. In the hope of developing an immunosensor for measurement of ALT activity, we have generated monoclonal antibodies to human recombinant ALT and fabricated them for use in a sensor. The ALT immunosensor was composed of the followings: (1) anti-ALT antibody-immobilized outer membrane; (2) pyruvate oxidase-absorbed inner membrane; (3) a self assembled monolayer mediator-coated gold working electrode and an Ag/AgCl reference electrode. The chronoamperometric measurement of the immunosensor was performed with 40 microl of PBS containing substrates and ALT without a washing step in less than 5 min. The dynamic range of ALT immunosensor was presented as five orders of magnitude, ranging between 10 pg/ml and 1 microg/ml. The detection limit and the sensitivity were 10 pg/ml and 26.3 nA/(ng/ml), respectively. In the meantime, the enzyme sensor fabricated without anti-ALT antibody showed much poorer analytical values. The dynamic range, the detection limit, and the sensitivity were 10 ng/ml-100 microg/ml, 10 ng/ml and 11.4 nA/(ng/ml), respectively. The presented results indicated that the immunosensor system provided much better technical performance in all of the aspects evaluated than did the enzyme sensor without the immobilized-antibody.