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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 ? uorescent 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 signi? cant 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 speci? c 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: [emailprotected] uoc. gr (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 speci? c 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 biosen sors 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) 384–388 385 formed with glucose oxidase (Taylor et al. , 1997; Kaszuba and Jones, 1999) on screen printed electrodes (Memoli et al. , 2002) as well as on chitosan gel beads (Wang et al. , 2003). In this study, the development of nano-biosensors using a different approach is presented. Our approach is based on the encapsulation of all the active reagents of an optical biosensor within the liposome environment. The analyte transport is achieved using porins which have been incorporated within the lipid bilayers. Based on the stabilizing effect of liposomes, the inherently unstable enzyme acetylcholinesterase from Drosophila melanogaster is encapsulated in the internal aqueous phase of liposomes. In order to have a sensitive transduction method through the walls of the liposome, a pH sensitive ? uorescence probe is used. These nanostructures are stable enough to be evaluated either as stand-alone nano-biosensors, or immobilized into an optimised sol–gel matrix. tometer (UNICAM 8625). Acryl optical cuvettes were obtained from Sarstedt. 2. 2. Liposome biosensor Encapsulation of acetylcholinesterase in egg phosphatidylcholine liposomes was performed following the lipid ? lm’s hydration technique (Chaize et al. , 2003). For the preparation of liposomes 5 mg egg phosphatidylcholine were mixed with 5 L of a 2 mg/mL stock solution of OmpF porin in 1% octyl-POE detergent. Then 100 L phosphate buffer 25 mM, pH = 7. 8 were added slowly to the solution and consequently 100 L of Drosophila AChE (? 500 OD/mL) and 30 L of 7. 6 ? 10? 5 M pyranine solution were introduced. The mixture was subjected to 50 freeze-thaw cycles by successive dipping in a liquid nitrogen bath (? 95 ? C) and a water bath (37 ? C). It has been reported in the literature that encapsulation ef? ciency increases by increasing number of freeze-thaw cycles (Chaize et al. , 2003; Colletier et al. , 2002). Even though the encapsulation ef? ciency is quite high even after only 10 freeze-thaw cycles, 50 cycles were chosen in order to obtain the maximum attainabl e encapsulation of enzyme in the liposomes. Next the sample was diluted to 1 mL with 25 mM phosphate buffer pH = 7. 8. To obtain liposomes of homogenous size the sample was extruded 10 times through polycarbonate ? lters with a pore size of 0. 2 m. The size of the liposome was measured to be 300  ± 4 nm in diameter using dynamic light scattering. The non-encapsulated enzyme was deactivated by adding 5 mg/mL pronase and incubating for 3 h at room temperature. In all cases, AChE activity was measured photometrically at 412 nm according to the sensitive Ellman’s method (Ellman et al. , 1961), in 25 mM phosphate buffer, pH = 7. 0 at 25 ? C. The schematic diagram for the development of the liposome biosensor is shown in Fig. 1. 2. 3. Liposome entrapment in sol–gel A silica sol was prepared by sonication for 20 min of TMOS/deionised water (5/1) in 0. 4 M HCl. The liposomes with the encapsulated enzyme were then mixed with the sol–gel solution at a ratio of 2:1, and the mixture was poured on the side of cuvettes. The immobilized enzyme in each cuvette was 6. 4 pmol. Gelation was occurred within a few minutes, resulting in a transparent ? lm. To avoid the shrinkage, 2. Materials and methods 2. 1. Reagents and ins trumentation Truncated cDNA encoding soluble wild type acetylcholinesterase from D. melanogaster (Dm. AChE), was expressed with the baculovirus system (Chaabihi et al. , 1994). Secreted AChE was puri? ed to homogeneity by af? ity chromatography as previously reported (Estrada-Mondaca and Fournier, 1998). Under these conditions the enzyme is stable for more than 1 week at room temperature. Expression and puri? cation of the outer membrane protein OmpF, from Escherichia coli has recently been described (Saint et al. , 1996). Acetylthiocholine chloride (ATChCl) and L-a phosphatidylcholine from egg yolk and the ? uorescent indicator pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid) were purchased from Sigma. Tetramethoxysilane (TMOS) was obtained from Aldrich. In all experiments nanopure water (? 18 M , EASYpure model D7033, Barnstead) was used. All other reagents used were of analytical grade. Fluorescence spectral data were recorded on an AmincoBowman series 2 luminescence spectrometer equipped with a continuous high power xenon lamp. All samples were analyzed at room temperature. The excitation and emission slits were set at 4 nm band-pass with a scan rate of 3 nm/s. Optical measurements were performed with a UV–vis spectropho- Fig. 1. Schematic representation of the liposome-biosensor design. 386 V. Vamvakaki et al. / Biosensors and Bioelectronics 21 (2005) 384–388 the cuvettes with the sol–gel were stored at 4 ? C in phosphate buffer 25 mM, pH = 7. 8. 2. 4. Measuring procedure All measurements were performed by adding the substrate acetylthiocholine chloride to the cuvettes containing the solution of AChE/liposomes or the immobilized AChE/liposomes in the sol–gel matrix. The porins embedded on the liposome membranes permit the substrate’s entrance in the internal nano-environment of the liposomes where the enzymatic reaction takes place. The latter results in the production of acetic acid and thus pH changes at the local nano-environment of the enzyme. This in turn alters the ? uorescence signal of the pyranine indicator which is subsequently correlated to the substrate concentration. Fig. 2. pH titration curve of pyranine. The change in ? uorescence signal of pyranine at 513 nm is recorded at different pH values. 3. Results and discussion The enzymatic reaction of AChE is shown in the following scheme: Acetylthiocholine + H2 O ? Thiocholine + Acetic acid Acetic acid production during the hydrolysis of the substrate acetylthiocholine results in the decrease of the pH value of the immediate aqueous environment surrounding the enzyme. AChE activity inside liposome is usually monitored using the classic photometric Ellman method (Chaize et al. , 2003; Ellman et al. , 1961). In this study a ? uorescence detection scheme is chosen as the transduction method. It is well known that ? uorescence provides higher sensitivity, lower detection limits and wider concentration range. In the proposed biosensor system, the ? uorescence detection of the enzymatic activity is accomplished simply by the introduction of a ? uorescent indicator within the liposome. This one-step procedure provides a simple self-contained stable nano-biosensor, thus avoiding the two steps required by the Ellman’s method for the detection of the enzymatic activity. AChE achieves optimum catalytic ef? iency when placed in neutral pH aqueous solutions. For this reason, and in order to develop a ? uorescent biosensor based on AChE, a pH indicator with pKa value close to pH 7. 0 is required. After a comprehensive literature search it became evident that Pyranine could ful? l these requirements. Pyranine is a pH sensitive ? uorescent indicator whose excitation and emission wavelengths wer e found at 460 and 513 nm, respectively. This indicator has a pKa value of 7. 3, it is highly soluble in water and membrane impermeable (Zignani et al. , 2000). Based on these characteristics, it is expected that this indicator could ful? the requirements set above for the optical AChE-based biosensor. In order to determine the working pH range, titration of the ? uorescent indicator was initially performed. As it can be seen from the resulting titration curve (Fig. 2) there is AChE a linear relationship between the pH value of the solution and the ? uorescence signal of the pyranine for a pH range between 7. 0 and 7. 8. In order to ensure the highest possible sensitivity of ? uorescence changes upon pH changes within the liposome, together with a wide dynamic range, the pH value of 7. 8 is chosen as the starting point. The time required to obtain a signi? cantly high signal for analytical purposes was determined next. To a buffered solution (pH 7. 8) containing the pH indicator pyranine and the enzyme AChE, additions of the substrate acetylthiocholine to different ? nal concentrations were performed. From these data (data not given) it was determined that the ? uorescent signal of the solution changed dramatically within a few minutes after substrate addition, and thus measurements could be made anytime after an initial 5 min waiting period. In the next step, the response of the liposome biosensor to acetylthiocholine was evaluated. The substrate was added to the enzyme loaded liposomes and the ? uorescent signal of pyranine was monitored over time. As shown in Fig. 3a the ? uorescence intensity of the indicator within the liposomes decreases with a rate that is proportional to the substrate concentration in the solution. Increasing the substrate concentration decreases the ? uorescence, with a response time in the order of a few minutes. Fig. 3b shows the calibration curve of the biosensor plotted as the ? uorescence intensity versus substrate concentration for 10 min reaction time. From this graph the substrate sensitivity of the sensor for substrate concentrations between 1. 0 and 13. 3 mM is calculated to be 8. 2 ? 10? 3 Abs min? 1 mM? 1 . In addition the detection limit of the sensor is calculated to be less than 1. 0 mM. Liposomes are very small, and thus dif? cult to handle as biosensors. In order to obtain a stand-alone biosensor device the biosensing element has to be immobilized on a more convenient platform. Such immobilization of the biosensor system can be used continuously and for successive measurements, while it can easily be introduced into other analytical systems, such as micro-devices or ? w injection systems. For this study, a transparent sol–gel matrix is used as the immobi- V. Vamvakaki et al. / Biosensors and Bioelectronics 21 (2005) 384–388 387 Fig. 3. (a) Fluorescence signal of AChE/liposome biosensor over time, for different ATChCl concentrations: 2. 5, 5. 0, 10. 0 and 13. 3 mM. (b) Calibration curve of the AChE/liposome b iosensor. To obtain the calibration curve, the ? uorescence intensity after 10 min reaction time was recorded for each substrate concentration. The immobilized enzyme was 6. 4 pmol and the ? uorescence signal was monitored at 513 nm. ization matrix of the nano-biosensor. The procedure for the formation of the sol–gel was precisely optimized in order to provide well-formed, smooth and reproducible membranes. For this reason, different sol–gel matrices were prepared and the most suitable for the speci? c use was chosen on the basis of ? lm quality (smooth ? lm, crack-free), transparency, and good adhesion to the polymeric optical window of the cuvette. The sol–gel biosensor was evaluated based on the ? uorescence signal of pyranine. The change of the biosensor signal with time at different substrate concentrations is shown in Fig. a, while the corresponding calibration curve obtained 10 min after the introduction of the substrate is shown in Fig. 4b. The biosensor showed a linear range of response that extended from 1. 0 to 15. 0 mM and the observed sensitivity to the substrate was 7. 5 ? 10? 3 Abs min? 1 mM? 1 . The sensorto-sensor reproducibility was very good with a R. S. D. value of less than 1% (N = 3). When the enzyme loaded liposomes are incubated with Triton X-100 the sensitivity of the biosensor increases on account of the lysis of the liposomes. This observation veri? es that the nzyme is encapsulated in the internal microenvironment of the liposome. It is also important to note that even the very small amount of enzyme incorporated in each biosensor system (6. 4 pmol) is suf? cient to obtain a reliable ? uorescence signal. This implies that using this biosensor system, monitoring of inhibitors (e. g. organophosphorus pesticides) can be achieved with high sensitivity and very low detection limit. A comparison between the AChE/liposome biosensor and the AChE/liposome sol–gel biosensor shows that the two systems have similar a nalytical characteristics. In both cases the response time is less than 10 min while the sensitivity of the biosensor remained approximately the same. Enzyme kinetics were also unaffected by the sol–gel immobilization step. It has been calculated that the apparent Km value is close to 5. 5 mM in both cases. This fact further enhances the ap- Fig. 4. (a) Fluorescence signal of the AChE/liposome sol–gel biosensor with time, for different ATChCl concentrations: 1. 0, 5. 0, 16. 6 and 33. 3 mM. (b) Calibration curve of the AChE/liposome sol–gel biosensor. To obtain the calibration curve, the ? orescence intensity after 10 min reaction time was recorded for each substrate concentration. The immobilized enzyme was 6. 4 pmol and the ? uorescence signal was monitored at 513 nm. 388 V. Vamvakaki et al. / Biosensors and Bioelectronics 21 (2005) 384–388 Acknowledgments This work is being supported by the program â€Å"Iraklitos† of the Greek Ministry of Education and the European Commiss ion Program â€Å"GANANO† (Contract No. STREP 505641-1). We would also like to thank Prof. A. K. Rizos for the Dynamic Light Scattering measurement. References Chaabihi, H. , Fournier, D. , Fedon, Y. , Bossy, J. P. , Ravallec, M. Devauchelle, G. , C? rutti, M. , 1994. Biochemical characterization of e Drosophila melanogaster acetylcholinesterase expressed by recombinant baculoviruses. Biochem. Biophys. Res. Commun. 203, 734–742. Chaize, B. , Winterhalter, M. , Fournier, D. , 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: ef? ciency depends on interactions between protein and phospholipid bilayer. BMC Biotechnol. 2, 9. Ellman, G. L. , Courtney, K. D. , Andres Jr. , V. , Featherstone, R. M. , 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Estrada-Mondaca, S. , Fournier, D. , 1998. Stabilization of recombinant drosophila acetylcholinesterase. Prot. Expr. Purif. 12, 166–172. Han, X. , Li, G. , Li, K. , 1998. FTIR study of the thermal denaturation of a-actinin in its lipid-free and dioleoylphosphatidylglycerol-bound states and the central and N-terminal domains of a-actinin in D2 O. Biochemistry 37, 10730–10737. Kaszuba, M. , Jones, M. N. , 1999. Hydrogen peroxide production from reactive liposomes encapsulating enzymes. Biochimica et Biophysica Acta 1419, 221–228. Kulin, S. , Kishore, R. , Helmerson, K. , Locascio, L. , 2003. Optical manipulation and fusion of liposomes as microreactors. Langmuir 19 (20), 8206–8210. Memoli, A. , Annesini, M. C. , Mascini, M. , Papale, S. , Petralito, S. , 2002. A comparison between different immobilised glucoseoxidase-based electrodes. J. Pharm. Biomed. Anal. 29, 1045–1052. Nasseau, M. , Boublik, Y. , Meier, W. , Winterhalter, M. , Fournier, D. , 2001. Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation. Biotechnol. Bioeng. 75, 615–618. Saint, N. , Windmer, C. , Luckey, M. , Schirmer, T. , Rosenbuch, J. P. , 1996. Structural and functional characterization of OmpF porin mutants selected for larger pore size. J. Biol. Chem. 271, 20676–20680. Singh, A. K. , Harrison, S. H. , Schoeniger, J. S. , 2000. Gangliosides as receptors for biological toxins: development of sensitive ? uoroimmunoassays using ganglioside-bearing liposomes. Anal. Chem. 72 (24), 6019–6024. Taylor, M. A. , Jones, M. N. , Vadgama, P. M. , Higson, S. P. , 1997. The effect of lipid bilayer manipulation on the response of the glucose oxidaseliposome electrode. Biosens. Bioelectron. 12, 467–477. Walde, P. , Ichikawa, S. , 2001. Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomol. Eng. 18, 143–177. Wang, S. , Yoshimoto, M. , Fukunaga, K. , Nakao, K. , 2003. Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor. Biotechnol. Bioeng. 83, 444–453. Winterhalter, M. , Hilty, C. , Bezrukov, S. M. , Nardin, C. , Meier, W. , Fournier, D. , 2001. Controlling membrane permeability with bacterial porins: application to encapsulated enzymes. Talanta 55, 965–971. Woodle, M. C. , 995. Sterically stabilized liposome therapeutics. Adv. Drug Deliv. Rev. 16, 249–265. Zignani, M. , Drummond, D. C. , Meyer, 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 of the sol–gel AChE biosensor over time for 16. 6 mM ATChCl: ( ) sol–gel with free AChE, and ( ) sol–gel biosensor with liposome immobilized AChE. The total amount of immobilized enzyme in both cases with and without liposome was 6. 4 pmol and the ? uorescence signal was monitored at 513 nm. licability of the sol–gel immobilized liposome biosensor, since this matrix does not introduce any additional diffusion barriers and thus it does not have any effect on enzyme kinetics. 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 ? uorescent signal over time for a given substrate concentration of the free AChE sol–gel biosensor shows signi? cant 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, ? uorescent 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 signi? cant 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.