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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 the ability to control their physicochemical properties, make them very appealing for a wide range of applications (Walde and Ichikawa, 2001). The most widespread application of liposomes is as carriers of functional substances and drugs. Controlled release of these substances is achieved under specific chemical or physical conditions. However due to their unique physical and chemical properties, liposomes can be used in a variety of other applications. For example, it has been observed that enzymes ∗

Corresponding author. Tel.: +30 2810 393 618; fax: +30 2810 393 601. E-mail address: (N.A. Chaniotakis).

are considerably stabilized within the nano-environment of liposomes, since they are protected from unfolding and proteolysis. Liposomes can effectively protect enzymes from the aggression of external agents such as proteases (Winterhalter et al., 2001). In addition, enzymes entrapped in liposomes are stabilized against unfolding forces due to hydrophobic interactions between the enzyme and the liposome membrane (Han et al., 1998). One other important characteristic is that enzymes encapsulated inside liposomes retain their activity even at very low concentrations (Nasseau et al., 2001). At the same time liposomes are optically translucent, and can thus be used as optical sensor elements (Kulin et al., 2003; Singh et al., 2000). Combining these characteristics one can envision that under specific experimental conditions they can be used for the development of nano-sized optical biosensors. Despite the fact that liposomes seem to be very promising nanomaterials in biosensor design only few reports dealing with this issue exist in literature. Initial attempts to develop liposome-based electrochemical biosensors have been per-

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.10.028

V. Vamvakaki et al. / Biosensors and Bioelectronics 21 (2005) 384–388


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