An electrochemical immunosensor for milk progesterone using a continuous flow system |
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Authors: | R M Pemberton J P Hart T T Mottram |
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Institution: | a Faculty of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK b Silsoe Research Institute, Wrest Park, Silsoe, Bedford MK45 4HS, UK |
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Abstract: | 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 (Eapp=+0.3 V vs. Ag/AgCl) over the range 0.1–1.0 μg/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 (imax, 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. |
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Keywords: | Progesterone Milk Screen-printed carbon electrode Continuous-flow |
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