Enzyme microspheres for biosensing and cell signaling perturbation
Concentration of lactate in human blood or saliva is an important indicator for cardiac diseases. Conventional measurement of analytes in complex solution usually requires separation, which is time consuming. To bypass these problems, we developed enzyme based sensor arrays that rapidly and simultaneously analyze multiple analytes in solutions without separation. In our approach, porous agarose beads, with enzymes for L-lactate immobilized on them, were localized to wells etched into a silicon chip in a 5 + 7 array. Analysis was accomplished by measuring fluorescence signal difference generated by enzymes catalyzed reactions. In this thesis, I reported the development of a sensitive (detection limit [similar to] 5[Greek small letter mu]M) and rapid (within minutes) lactate assay using double-enzyme based sensor array. Lactate concentration in healthy people at rest was not detected partially due to enzyme inhibitions by protein adsorptions. Measurement of enzyme activity on individual beads is another research performed in this thesis. Success in this study should provide a more precise control over site-specific cellular manipulation by delivering a particle, decorated with optimal amount of effectors, into specific location of a living cell using optical trapping. In our approach, alkaline phosphatase (ALP) was used as an effector to generate product emitting fluorescence signal. Enzyme activity on individual microsphere was evaluated by signal strength. Separation of a single or small number of beads was accomplished using methods based on microfabrication via multiphoton excitation. The fluorescence signal was not detected on single beads partially due to the enzyme inhibition due to BSA quenching.