Biosensors and Bioelectronics twenty one (2005) 384–388

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Fluorescence detection of enzymatic activity in a liposome centered nano-biosensor Vicky Vamvakakia, Didier Fournierb, Nikos A. Chaniotakisa, ∗ a

Laboratory of Analytical Chemistry, Department of Chemistry, Knossou Avenue, College or university of Crete, 71409 Iraklion, Crete, Greece b IPBS, 205 Way de Narbonne, 31077 Toulouse, France Received 26 Come july 1st 2004; received in modified form twenty-two September 2005; accepted twenty-five October 2005 Available online almost 8 December 2004

Abstract The encapsulation of enzymes in microenvironments and especially in liposomes, has which may greatly boost enzyme leveling against unfolding, denaturation and dilution results. Combining this kind of stabilization effect, with the reality liposomes will be optically clear, we have designed nano-sized circular biosensors. Through this work liposome-based biosensors have decided by encapsulating the chemical acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with the average diameter of 300 ± 4 nm. Porins are embedded in to the lipid membrane, allowing for the free substrate transport, but is not that of the enzyme due to size limits. The enzyme activity inside the liposome is usually monitored employing pyranine, a fluorescent pH indicator. The response from the liposome biosensor to the base acetylthiocholine chloride is relatively fast and reproducible, while the strategy is stable since has been shown by simply immobilization within sol–gel. © 2004 Elsevier B. Sixth is v. All privileges reserved. Keywords: Encapsulation; Liposomes; Fluorescent probe; Biosensor; Acetylcholinesterase

1 . Introduction Liposomes are nanoscale spherical shells consisting of lipid bilayers that block off an aqueous phase. They are easily developed and steady in answer for a long period of time, with no significant changes in size or framework (Woodle, 1995). In addition the biocompatible microenvironment of the liposomes, along with the capacity to control their very own physicochemical properties, make them very appealing for any wide range of applications (Walde and Ichikawa, 2001). The most popular application of liposomes is as service providers of efficient substances and drugs. Controlled launch of these substances is attained under specific chemical or physical conditions. However due to their one of a kind physical and chemical real estate, liposomes can be used in a variety of various other applications. For example , it has been discovered that digestive enzymes ∗

Corresponding author. Tel.: +30 2810 393 618; fax: +30 2810 393 601. E-mail address: [email protected] uoc. grms (N. A. Chaniotakis).

happen to be considerably stable within the nano-environment of liposomes, since they are guarded from unfolding and proteolysis. Liposomes can effectively protect enzymes from the aggression of external agents such as proteases (Winterhalter ainsi que al., 2001). In addition , digestive enzymes entrapped in liposomes are stabilized against unfolding causes due to hydrophobic interactions involving the enzyme and the liposome membrane layer (Han ainsi que al., 1998). One other significant characteristic is the fact enzymes encapsulated inside liposomes retain their very own activity also at very low concentrations (Nasseau et al., 2001). At the same time liposomes are optically clear, and can thus be used while optical sensor elements (Kulin ain al., 2003; Singh ainsi que al., 2000). Combining these kinds of characteristics one can possibly envision that under specific experimental conditions they can be utilized for the development of nano-sized optical biosensors. Despite the fact that liposomes seem to be extremely promising nanomaterials in biosensor design just few reports dealing with this problem exist in literature. Preliminary attempts to formulate liposome-based electrochemical biosensors have been per-

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Referrals: Chaabihi, H., Fournier, D., Fedon, Y., Bossy, L. P., Ravallec, M., Devauchelle, G., C´ rutti, M., 1994. Biochemical characterization of e Drosophila melanogaster acetylcholinesterase expressed by simply recombinant baculoviruses. Biochem. Biophys. Res. Modere. 203, 734–742. Chaize, N., Winterhalter, Meters., Fournier, M., 2003. Encapsulation of acetylcholinesterase in preformed liposomes. BioTechniques 34, 1158–1162. Colletier, J. P., Chaize, B., Winterhalter, M., Fournier, D., 2002. Protein encapsulation in liposomes: efficiency depends upon interactions among protein and phospholipid bilayer. BMC Biotechnol. 2, on the lookout for. Ellman, G. L., Courtney, K. Deb., Andres Junior., V., Featherstone, R. Meters., 1961. A new and quick colorimetric perseverance of acetylcholinesterase activity. Biochem. Pharmacol. six, 88–95. Estrada-Mondaca, S., Fournier, D., 1998. Stabilization of recombinant drosophila acetylcholinesterase. Prot. Expr. Purif. 12, 166–172. Han, Back button., Li, G., Li, K., 1998. FTIR study of the thermal denaturation of a-actinin in its lipid-free and dioleoylphosphatidylglycerol-bound states plus the central and N-terminal websites of a-actinin in D2 O. Biochemistry and biology 37, 10730–10737. Kaszuba, Meters., Jones, Meters. N., 1999. Hydrogen peroxide production coming from reactive liposomes encapsulating digestive enzymes. Biochimica ainsi que Biophysica Rese?a 1419, 221–228. Kulin, T., Kishore, R., Helmerson, K., Locascio, T., 2003. Optical manipulation and fusion of liposomes as microreactors. Langmuir 19 (20), 8206–8210. Memoli, A., Annesini, M. C., Mascini, Meters., Papale, S i9000., Petralito, S i9000., 2002. A comparison between several immobilised glucoseoxidase-based electrodes. M. Pharm. Biomed. Anal. 29, 1045–1052. Nasseau, M., Boublik, Y., Meier, W., Winterhalter, M., Fournier, D., 2001. Substrate-permeable encapsulation of enzymes maintains powerful activity, stabilizes against denaturation, and helps to protect against proteolytic degradation. Biotechnol. Bioeng. 75, 615–618. St ., N., Windmer, C., Luckey, M., Schirmer, T., Rosenbuch, J. G., 1996. Strength and useful characterization of OmpF porin mutants selected for greater pore size. J. Biol. Chem. 271, 20676–20680. Singh, A. E., Harrison, T. H., Schoeniger, J. T., 2000. Gangliosides as pain for neurological toxins: development of sensitive fluoroimmunoassays using ganglioside-bearing liposomes. Anal. Chem. 72 (24), 6019–6024. Taylor, M. A., Smith, M. In., Vadgama, G. M., Higson, S. G., 1997. The effect of lipid bilayer manipulation on the response of the glucose oxidaseliposome electrode. Biosens. Bioelectron. 12, 467–477. Walde, L., Ichikawa, H., 2001. Nutrients inside lipid vesicles: preparing, reactivity and applications. Biomol. Eng. 18, 143–177. Wang, S., Yoshimoto, M., Fukunaga, K., Nakao, K., the year 2003. Optimal covalent immobilization of glucose oxidase-containing liposomes pertaining to highly stable biocatalyst in bioreactor. Biotechnol. Bioeng. 83, 444–453. Winterhalter, M., Hilty, C., Bezrukov, S. Meters., Nardin, C., Meier, T., Fournier, Deb., 2001. Managing membrane permeability with microbial porins: software to encapsulated enzymes. Talanta 55, 965–971. Woodle, M. C., 95. Sterically stable liposome therapeutics. Adv. Medicine Deliv. Revolution. 16, 249–265. Zignani, M., Drummond, G. C., She, O., Hong, K., Leroux, J. C., 2000. In vitro characterization of a novel polymeric-based pH-sensitive liposome system. Biochim. Biophys. Acta 1463, 383–394.

Fig. 5. Fluorescence signal in the sol–gel AChE biosensor as time passes for of sixteen. 6 mM ATChCl: ( ) sol–gel with free AChE, and ( ) sol–gel biosensor with liposome immobilized Soreness. The total amount of immobilized enzyme in both equally cases with and without liposome was 6. 4 pmol and the fluorescence signal was monitored in 513 nm.

plicability with the sol–gel immobilized liposome biosensor, since this matrix does not expose any additional durchmischung barriers and therefore it does not have any effect on chemical kinetics. Considering that the by-products in the sol–gel process can be bad for the digestive enzymes, sol–gel biosensors with cost-free AChE and liposome crammed AChE were prepared and evaluated. As they can be seen via Fig. 5 the fluorescent signal over time for a given substrate attention of the free of charge AChE sol–gel biosensor displays significant destruction on the sensitivity over time, when compared to biosensor with liposome immobilized AChE. This reduced response of the cost-free AChE biosensor, versus the liposome based you are attributed to partial deactivation in the AChE in the sol–gel matrix. The stability from the liposome immobilized AChE biosensor indicates that the enzyme is definitely considerably stabilized against denaturation from the methanol produced during the hydrolysis technique of the silicate solution.

four. Conclusions With this paper a novel biosensor system originated using porin embedded AChE loaded liposomes containing pyranine as the optical, fluorescent indicator. The nano-sized liposomes provide a suitable environment intended for the effective stabilization of enzymes. The porins permit the expedient transfer of the substrate through the liposome walls, as the enzyme is entrapped because of physical size. The incorporation of these enzyme loaded liposomes into sol–gel matrices provides an optically energetic biosensor with good overall analytical features. The proven ability to screen very low enzymatic activity, the actual good sensor-to-sensor reproducibility as well as the significant stableness of the program provide the environment for the application of the provided nanobiosensors in the detection of organophosphorus pesticides and other harmful AChE inhibitors.

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