Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.
The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.
Vamvakaki
Vicky
V
Laboratory of Analytical Chemistry, Department of Chemistry, Knossou Avenue, University of Crete, 71409 Iraklion, Crete, Greece.
Fournier
Didier
D
Chaniotakis
Nikos A
NA
D000110:Acetylcholinesterase / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002800:Cholinesterase Inhibitors / Q000032:analysis* / Q000737:chemistry*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008081:Liposomes / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods
D002800:Cholinesterase Inhibitors; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D008081:Liposomes; D000110:Acetylcholinesterase