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 conditions. Single-use biosensors were fabricated by depositing anti-progesterone 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) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E(app)=+0.3 V vs. Ag/AgCl) over the range 0.1-1.0 microg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate concentration=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i(max), nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0-50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0-25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.
Pemberton
R M
RM
Faculty of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK.
Hart
J P
JP
Mottram
T T
TT
D000818:Animals; D000911:Antibodies, Monoclonal; D015374:Biosensing Techniques / Q000295:instrumentation*; D002417:Cattle; D004563:Electrochemistry; D007118:Immunoassay / Q000295:instrumentation; D008892:Milk / Q000737:chemistry*; D009284:Naphthols; D011374:Progesterone / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D009284:Naphthols; C029350:1-naphthol; D011374:Progesterone