Biosensors and Bioelectronics: A Biosensor System Made of Nano-Sized Liposomes

Biosensors and Bioelectronics 21 (2005) 384–388

Short communication

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor
Vicky Vamvakakia , Didier Fournierb , Nikos A. Chaniotakisa,∗ a Laboratory of Analytical Chemistry, Department of Chemistry, Knossou Avenue, University of Crete, 71409 Iraklion, Crete, Greece b IPBS, 205 Route de Narbonne, 31077 Toulouse, France Received 26 July 2004; received in revised form 22 September 2004; accepted 25 October 2004 Available online 8 December 2004

Abstract 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. © 2004 Elsevier B.V. All rights reserved.
Keywords: Encapsulation; Liposomes; Fluorescent probe; Biosensor; Acetylcholinesterase

1. Introduction Liposomes are nanoscale spherical shells composed of lipid bilayers that enclose an aqueous phase. They are easily produced and stable in solution for a long period of time, with no significant changes in size or structure (Woodle, 1995). In addition the biocompatible microenvironment of the liposomes, along with

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Since the by-products of the sol–gel process can be detrimental to the enzymes, sol–gel biosensors with free AChE and liposome loaded AChE were prepared and evaluated. As it can be seen from Fig. 5 the fluorescent signal over time for a given substrate concentration of the free AChE sol–gel biosensor shows significant deterioration on the sensitivity over time, compared to the biosensor with liposome immobilized AChE. This reduced response of the free AChE biosensor, versus the liposome based one is attributed to partial deactivation of the AChE in the sol–gel matrix. The stability of the liposome immobilized AChE biosensor indicates that the enzyme is considerably stabilized against denaturation from the methanol produced during the hydrolysis process of the silicate solution. 4. Conclusions In this paper a novel biosensor system was developed using porin embedded AChE loaded liposomes containing pyranine as the optical, fluorescent indicator. The nano-sized liposomes provide a suitable environment for the effective stabilization of enzymes. The porins allow for the expedient transport of the substrate through the liposome walls, while the enzyme is entrapped due to its physical size. The incorporation of these enzyme loaded liposomes into sol–gel matrices provides an optically active biosensor with good overall analytical characteristics. The proven ability to monitor very low enzymatic activity, the very good sensor-to-sensor reproducibility and the significant stability of the system provide the grounds for the application of the presented nanobiosensors in the detection of organophosphorus pesticides and other toxic AChE inhibitors.