RESUMEN
Mastitis, an inflammation of the mammary gland and udder tissue, is the major endemic disease of dairy cattle. In addition to causing health problems to the animals, mastitis leads to the reduction of milk production and quality, representing a significant economic burden for farmers. To enable timely treatment of infected animals with pathogen-specific antibiotics, the development of automated analytical methods for rapid on-site identification and quantification of mastitis-causing pathogens in milk is particularly important. An immunosensing system for multiplex detection of the two most common mastitis-causing pathogens Staphylococcus aureus and Escherichia coli is proposed in the present study. This immunosensor combines Bead Injection Analysis (BIA), attachment of pathogens onto renewable micro-column, biorecognition of bound pathogens by specific antibodies, conjugated with different fluorescence markers and the measurement of fluorescence signals. The analysis takes 20min and exhibits detection limits of < 50 CFU mL-1 for E. coli and 100 CFU mL-1 for S. aureus in milk. The applicability of the immunosensor was demonstrated by analyzing milk samples from cows, who were suffering from acute clinical mastitis.
Asunto(s)
Escherichia coli/aislamiento & purificación , Inmunoensayo/métodos , Mastitis Bovina/microbiología , Leche/microbiología , Staphylococcus aureus/aislamiento & purificación , Animales , Bovinos , Escherichia coli/fisiología , Femenino , Humanos , Límite de Detección , Staphylococcus aureus/fisiologíaRESUMEN
We studied the modulation of calibration parameters of biosensors, in which glucose oxidase was used for bio-recognition, in the presence of different chlorides by following the transient phase dynamics of oxygen concentration with an oxygen optrode. The mechanism of modulation was characterized with the changes of the glucose oxidase catalytic constant and oxygen diffusion constant. The modulation of two biosensor calibration parameters were studied: the maximum calculated signal change was amplified for about 20% in the presence of sodium and magnesium chlorides; the value of the kinetic parameter decreased along with the addition of salts and increased only at sodium chloride concentrations over 0.5 mM. Besides glucose bioassay, the amplification of calibration parameters was also studied in cascaded two-enzyme lactose biosensor, where the initial step of lactose bio-recognition, the ß-galactosidase - catalyzed lactose hydrolysis, was additionally accelerated by magnesium ions.