Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk

A total internal reflectance fluorescence (TIRF)-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples for 25 days, covering a whole estrus cycle. The detection is based on total internal reflectance fluorescence. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal anti-progesterone antibody as biological recognition element. First an existing progesterone assay was optimized by reducing the assay time per measurement, resulting in an assay time of about 5 min and reaching a limit of detection (LOD) of 0.04 ng mL(-1) and a quantification limit (LOQ) of 0.34 ng mL(-1). After calibration the assay was tested by measuring the progesterone level in daily milk samples over several weeks. An estrus cycle of a cow could be measured. As results become available within minutes without any preparation or pre-concentration of the milk samples the fully automated TIRF-based biosensor for progesterone can be used in-line in the milking parlor and thus could be an important tool for reproductive management of dairy cattle detecting heat and predicting pregnancy, which are critical parameters in milk production. (c) 2006 Elsevier B.V. All rights reserved.