My thesis is related to designing and implementing a Lab-on-a-Chip (LoC) device that in the future may provide an opportunity to diagnose a genetic disease, phenylketonuria (PKU).
The function of the chip is based on athe ammonia elimination of L-phenylalanine catalyzed by phenylalanine ammonia-lyase (PAL). The PAL enzyme molecules are immobilized on the surface of magnetic nanoparticles (MNPS), then as a magnetic fluid is transferred into the chip. We investigated how the various reaction conditions affect the activity of these immobilized enzyme molecules
MNPs are increasingly used in biomedicine both in vitro and in vivo. Because of their magnetic properties they are suitable for MRI contrast agent, drug carriers in targeted therapy and tumor diseases therapy (hyperthermia therapy). The magnetic nanoparticles are extremely popular in microfluidics, too. They are generally used in molecular biology and immunoassays, applied for separation of the individual reaction components or carrier.
Both free and immobilized PAL enzyme activity were tested in the elimination of L-phenylalanine, where cinnamic acid is formed. The absorbance of the generated cinnamic acid can be measured with UV-VIS spectrophotometer. Spectrophotometric measurements were performed after calibration to test trapped MNP-PAL activity. The temperature dependence of free and immobilized PAL was compared and catalitic activity in presence of polyethylene glycol (PEG) (different molecular weights and amounts) was also investigated. Finally, we performed measurements in LoC environment according to the previous results.