An electrochemical immunosensor for milk progesterone using a continuous flow system

An electrochemical biosensor for cow's milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow co nditions. Single-use biosensors were fabricated by depositing anti-progeste rone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs ). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixt ure, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substr ate (1-naphthyl phosphate, I-NP). In the thin-layer cell, the enzyme produc t, 1-naphthol, showed electrochemical behaviour consistent with bulk condit ions and gave a linear amperometric response under continuous-flow conditio ns (E-app = +0.3 V vs. Ag/AgCl) over the range 0.1-1.0 mug/ml. After pre-in cubating biosensors with progesterone standards, signal generation within t he cell (substrate concentration = 5 mM) was recorded amperometrically as r ate (nA/s) or maximum current (i(max), nA). Response values for milk standa rds were approximately 50% of those prepared in buffer. In both cases, cali bration plots over the range 0-50 ng/ml progesterone were obtained. By cond ucting sample binding under flowing conditions, only 7% of the previous res ponse was obtained, even at a substrate concentration of 50 mM, resulting i n low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sam ple binding, followed by continuous flow), low-noise amperograms were obtai ned at [1-NP] = 5 mM. Calibration plots were obtained over the range 0-25 n g/ml, with a coefficient of variation of 12.5% for five replicate real milk samples. (C) 2001 Elsevier Science BN. All rights reserved.