Polyelectrolyte-Mediated Assembly of Multiwalled Carbon Nanotubes

Nov 9, 2009 - Thanh Dat Nguyen , Stéphane Guyot , Jeannine Lherminier , Yves Wache , Rémi Saurel , Florence Husson. Process Biochemistry 2015 50 (10...
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Polyelectrolyte-Mediated Assembly of Multiwalled Carbon Nanotubes on Living Yeast Cells Alsu I. Zamaleeva,† Ilziya R. Sharipova,† Anna V. Porfireva,‡ Gennady A. Evtugyn,‡ and Rawil F. Fakhrullin*,† †

Department of Biochemistry and ‡Department of Analytical Chemistry, Kazan State University, Kreml uramı 18, Kazan, 420008, Tatarstan, Russian Federation Received August 7, 2009. Revised Manuscript Received September 23, 2009

Here we report the three-dimensional assembly of carbon nanotubes on the polyelectrolyte-coated living Saccharomyces cerevisiae cells using the polyelectrolyte-mediated layer-by-layer approach. Synthetic polyelectrolytes poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) were layer-by-layer deposited on the surfaces of the yeast cells followed by the deposition of water-soluble oxidized multiwalled carbon nanotubes (MWNTs) and an additional outermost polyelectrolyte bilayer. This resulted in the fabrication of polyelectrolyte/nanotubes composite coatings on the cell walls of the yeast cells, which could be clearly seen using the conventional optical microscopy. Transmission and scanning electron microscopy was applied to further investigate the composite coatings. Viability of the encapsulated cells was confirmed using the intercellular esterase activity test. Finally, electrochemical studies using voltammetry and electrochemical impedance measurements were performed, indicating that the composite polyelectrolytes/MWNTs coatings sufficiently affect the electron mediation between the encapsulated yeast cells and the artificial electron acceptor, making it possible to distinguish between living and dead cells. The technique described here may find potential application in the development of microelectronic devices, core-shell and hollow composite microparticles, and electrochemical cell-based biosensors.

Introduction Nature offers us an enormous amount of ready-to-use templates with various morphologies and functionalities, which can be successfully utilized in fabrication of biosensors, novel catalysts, tissue engineering, and microelectronics. The directed combination of natural templates with inorganic nanomaterials results in the development of novel methods and devices which unite the useful features of both.1 Unicellular microorganisms, such as bacteria, fungi, and algae, are extensively used in biotechnology and life sciences as simple yet functional model species, catalysts, genetically modified organisms, and so forth. Well-defined monodisperse sizes and shapes as well as the lowcost and scalable grow of microbial cells are the key reasons to use them in such applications as the development of novel materials and hybrid microdevices, which utilize the features of living cells along with characteristics of inorganic and/or organic elements (nanoparticles, nanofibers, multilayer coatings, etc.). Recently, living microbial cells have attracted considerable interest as the templates for the direct deposition of inorganic nanoparticles2 and functionalized nanorods3 to produce novel microelectronic devices, in addition, the layer-by-layer (LbL) assembly of oppositely charged polymers on the surfaces of living yeast cells4 and mouse stem cells5 has been demonstrated. Microbial cells, having a variety of shapes and sizes, can be regarded as effective and cheap templates for the deposition of nanomaterials, allowing for *Corresponding author. Phone: þ78432337833; E-mail: kazanbio@gmail. com, [email protected]. (1) Fan, X.; Lei, J. Adv. Mater. 2008, 20, 2842–2858. (2) Berry, V.; Rangaswamy, S.; Saraf, R. F. Nano Lett. 2004, 4, 939–942. (3) Berry, V.; Gole, A.; Kundu, S.; Murphy, C. J.; Saraf, R. F. J. Am. Chem. Soc. 2005, 127, 17600–17601. (4) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047–5050. (5) Veerabadran, N. G.; Goli, P. L.; Stewart-Clark, S. S.; Lvov, Y. M.; Mills, D. M. Macromol. Biosci. 2007, 7, 877–882.

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scalable production of microelectronic devices such as electronic conductive microbridges2 and other hybrid cell-based microdevices. In fact, the diversity of microbial cell morphologies and the ability to select the required shapes or sizes of the cellular templates for the further surface modification with functional nanomaterials allows for the fabrication of an almost unlimited variety of resulting cell-based microdevices. Currently, several approaches for the deposition of nanoparticles onto the surfaces of microbial cells exist, mainly employing the specific recognition of biopolymer-modified nanoparticles by cell wall macromolecules2 and via the direct adsorption of as-made nanoparticles.6 In addition, several microbial species were reported to reduce metal ions on the surfaces of cellular walls; therefore, an interesting approach for the modification of the living cells by in situ synthesis of nanoparticles on the bacterial cell walls was proposed.7 Polyelectrolyte nanofilms doped with various inorganic nanoparticles can also be deposited on the microbial and mammalian living cells. Blood cells (platelets) were layer-by-layer coated with polyelectrolytes and nanoparticles (silica and fluorescent latex) in order to modify such cellular properties as platelet aggregation and secretion; in addition, abnormal receptor-agonist interactions can be blocked by the composite polymer-nanoparticles coatings.8 Microbial cells were coated with consecutive layers of polymers and nanomaterials as well; for example, fungi cells (yeast cells and Trichoderma asperellum conidia) were coated with alternating layers of polyelectrolytes and noble metal nanoparticles.9 Just recently, a similar (6) Safarik, I.; Rego, L. F. T.; Borovska, M.; Mosiniewicz-Szablewska, E.; Weyda, F.; Safarikova, M. Enzyme Microb. Technol. 2007, 40, 1551–1556. (7) Kuo, W. S.; Wu, C. M.; Yang, Z. S.; Chen, S. Y.; Chen, C. Y.; Huang, C. C.; Li, W. M.; Sun, C. K.; Yeh, C. S. Chem. Commun. 2008, 37, 4430–4432. (8) Ai, H.; Fang, M.; Jones, S. A.; Lvov, Y. M. Biomacromolecules 2002, 3, 560– 564. (9) Fakhrullin, R. F.; Zamaleeva, A. I.; Morozov, M. V.; Tazetdinova, D. I.; Alimova, F. K.; Hilmutdinov, A. K.; Zhdanov, R. I.; Kahraman, M.; Culha, M. Langmuir 2009, 25, 4628–4634.

Published on Web 11/09/2009

DOI: 10.1021/la902937s

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approach was utilized for the deposition of silica nanoparticles on bacteria spores,10 the techniques described in the respective papers can be utilized in surface-enhanced Raman scattering studies of individual fungal cells and for the imaging of polyelectrolytecoated bacterial spores. Obviously, the surface modification of living microbial cells is a promising way for the development of novel cell-based hybrid microdevices and biomimetic materials; in addition, the interfacing of nanomaterials with living cells might be a versatile way to study the toxicity issues of nanomaterials. In recent years, carbon nanotubes, due to their unique physicochemical properties, have attracted significant interest and are currently used in many applications ranging from the development of novel materials to biomedical applications.11-14 It has been suggested that the spatial 2D and 3D arrangement of carbon nanotubes in relatively large multicomponent structures, such as multilayered coatings,15 free-standing thin films,16 or spherical hollow colloid particles (nanotubosomes) built either using emulsions17,18 or sacrificial microparticles,19 may be effective in the development of novel nanoscale electronic devices and encapsulation of catalysts. Just recently, a novel approach for the 3D arrangement of carbon nanotubes using lignocellulose wood microfibers as templates with the aim of producing conductive microfibers and paper sheets has been reported.20 The spatial arrangement of carbon nanotubes in hybrid multilayered complexes with biomacromolecules and nanoparticles is also employed in a number of bioelectronic devices, including electrophysiology instruments,21 electrochemical biosensors,22,23 and photochemical solar cells.24 In addition, the potential of carbon nanotubes incorporated in multilayered coatings and freestanding films in tissue engineering,25 stem cells differentiation,26 and the development of implantable devices27 was recently demonstrated. As a result, many efforts are currently focused on the development of novel techniques for functionalization of various substrates and templates with carbon nanotubes.28-30

(10) Balkundi, S. S.; Veerabadran, N. G.; Eby, D. M.; Johnson, G. R.; Lvov, Y. M. Langmuir 2009, doi:10.1021/la900971h. (11) Chen, X.; Tam, U. C.; Czlapinski, J. L.; Lee, G. S.; Rabuka, D.; Zettl, A.; Bertozzi, C. R. J. Am. Chem. Soc,. 2006, 128, 6292–6293. (12) Vakarelski, I. U.; Brown, S. C.; Higashitani, K.; Moudgil, B. M. Langmuir 2007, 23, 10893–10896. (13) Gjerde, K.; Kumar, R. T. R.; Andersen, K. N.; Kjelstrup-Hansen, J.; Teo, K. B. K.; Milne, W. I.; Persson, Ch.; Moelhave, K.; Rubahn, H.-G.; Boeggild, P. Soft Matter 2008, 4, 392–399. (14) Wang, Y.; Joshi, P. P.; Hobbs, K. L.; Johnson, M. B.; Schmidtke, D. W. Langmuir 2006, 22, 9776–9783. (15) Paloniemi, H.; Lukkarinen, M.; Aaritalo, T.; Areva, S.; Leiro, J.; Heinonen, M.; Haapakka, K.; Lukkari, J. Langmuir 2006, 22, 74–83. (16) Ko, H.; Jiang, Ch.; Shulha, H.; Tsukruk, V. V. Chem. Mater. 2005, 17, 2490–2493. (17) Haixia, Y.; Huaihe, S.; Xiaohong, C. Langmuir 2007, 23, 3199–3204. (18) Panhuis, M.; Paunov, V. N. Chem. Commun. 2005, 13, 1726–1728. (19) Paunov, V. N.; Panhuis, M. Nanotechnology 2005, 16, 1522–1525. (20) Agarwal, M.; Xing, Q.; Shim, B. S.; Kotov, N.; Varahramyan, K.; Lvov, Y. Nanotechnology 2009, 20, 215602. (21) Gheith, M. K.; Pappas, T. C.; Liopo, A. V.; Sinani, V. A.; Shim, B. S.; Motamedi, M.; Wicksted, J. P.; Kotov, N. A. Adv. Mater. 2006, 18, 2975–2979. (22) Zhao, L.; Liu, H.; Hu, N. Anal. Bioanal. Chem. 2006, 384, 414–422. (23) Zhang, J.; Feng, M.; Tachikawa, H. Biosens. Bioelectron. 2007, 22, 3036– 3041. (24) Hasobe, T.; Fukuzumi, S.; Kamat, P. V. J. Phys. Chem. 2006, 110, 25477– 25484. (25) Zhao, B.; Hu, H.; Mandal, S. K.; Haddon, R. C. Chem. Mater. 2005, 17, 3235–3241. (26) Jan, E.; Kotov, N. A. Nano Lett. 2007, 7, 1123–1128. (27) Gheith, M. K.; Sinani, V. A.; Wicksted, J. P.; Matts, R. L.; Kotov, N. A. Adv. Mater. 2005, 17, 2663–2670. (28) Firkowska, M. O.; Pazos-Perez, N.; Rojas-Chapana, J.; Giersig, M. Langmuir 2006, 22, 5427–5434. (29) Bekyarova, E.; Thostenson, E. T.; Yu, A.; Kim, H.; Gao, J.; Tang, J.; Hahn, H. T.; Chou, T.-W.; Itkis, M. E; Haddon, R. C. Langmuir 2007, 23, 3970–3974. (30) Havel, M.; Behler, K.; Korneva, G.; Gogotsi, Y. Adv. Funct. Mater. 2008, 18, 2322–2327.

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Obviously, the utilization of living cells as versatile templates for the deposition of carbon nanotubes to produce highly organized 3D hybrid microstructures offers many potential advantages, and the resulting nanotube-decorated cells may find a number of practical applications in bioelectronics and electrochemical biosensors. Although the previously reported methods17-19 are effective and lead to the formation of spherical hollow carbon nanotubes composite structures, they suffer from time-consuming steps and polydisperse size distribution of the resulting carbon nanotubes microcapsules. It is clear that the technique utilizing living cells as the templates for the deposition of carbon nanotubes would benefit from the almost unlimited diversity of biological cells. Previously, glycan-mimicking glycopolymers were used to functionalize carbon nanotubes and then to interface these modified nanotubes with Chinese hamster ovary cells.11 Although this resulted in the relatively effective coating of mammal cells with carbon nanotubes (therefore, making it possible to use this technique in biological processes probing, imaging, etc), this approach requires a sophisticated modification of nanotubes with a receptor-specific glycopolymer; therefore, it hardly can be used for a scalable modification of cells. Moreover, mammal cells are obviously poor templates because they lack a solid cell wall and require special growing conditions, which further increases the expenses of using mammal cells as templates. Herein, we report the assembly of multiwalled carbon nanotubes (MWNTs) on the surfaces of living Saccharomyces cerevisiae baker’s yeast cells using the LbL deposition of oppositely charged polyelectrolytes. The technique described uses the commercially available and relatively inexpensive synthetic polymers and can be applied to the microbial cells without any special surface modification of carbon nanotubes. Our approach is based on the immobilization of partially hydrophilic oxidized MWNTs onto the oppositely charged polyelectrolyte-coated yeast cells with the subsequent deposition of polyelectrolytes in order to “seal” the MWNTs on the cell walls. Using optical and electron microscopy, we demonstrate that the polyelectrolyte-mediated assembly results in the deposition of a dense monolayer of nanotubes on the cell surfaces. Importantly, the polyelectrolyte/MWNT-coated yeast cells maintain their viability, which is demonstrated using the fluorescence enzyme-activity test. Electrochemical investigations using direct current voltammetry and electrochemical impedance measurements indicate that the polyelectrolyte/MWNT coatings sufficiently affect the electron mediation between yeast cells and artificial electron acceptor. This can be further used for distinguishing living and dead cells in various tests related to the toxicity assessment or cell viability estimation.

Materials and Methods Chemicals. The following reagents were used in this study: poly(allylamine hydrochloride) (PAH, Mw ca. 15 000 Da), poly(fluorescein isothiocyanate allylamine hydrochloride) (FITCPAH, Mw ca. 15 000 Da), and poly(sodium 4-styrenesulfonate) (PSS, Mw ca. 70 000 Da) were purchased from Sigma-Aldrich, and fluorescein diacetate (FDA) was purchased from Fluka; these reagents were used as received. Multiwalled carbon nanotubes (MWNTs; outer diameter 10-30 nm, inner diameter 5-10 nm, length 0.5-500 μm, as provided by the supplier) were supplied by Aldrich. Other reagents were of analytical grade and used without purification. All experiments were carried out at room temperature; all solutions were prepared using Milli-Q water (Milli-Q Academic water purification setup). MWNTs Oxidation. The aqueous suspension of MWNTs was pretreated with 3 M HCl for 1 h at 60 °C while sonicating and then oxidized in the mixture of concentrated nitric (67 wt %) and sulfuric (98 wt %) acids 1:4 (v/v) under sonication for 3 h. Then, Langmuir 2010, 26(4), 2671–2679

Zamaleeva et al. the oxidized MWNTs were washed with water until the neutral pH was detected, separated from the aqueous phase by centrifugation, and dried at 80 °C. Prior to deposition on the surfaces of the yeast cells, the MWNTs were dispersed in water to final concentration of 1 mg mL-1 and sonicated for 15 min. The aqueous suspension of thus oxidized MWNTs was stable at least for 2 weeks. Yeast Cells. Baker’s yeast cells S. cerevisiae were purchased from a local store. Before coating, the cells were suspended in water and washed three times with water followed by centrifugation. Then, the aqueous suspensions of 10 mg mL-1 (wet weight) yeast cells were prepared and used for LbL coating with polyelectrolytes and MWNTs. In order to thermally inactivate the yeast cells, they were boiled in water for 2 h, then separated by centrifugation and washed with water.

LbL Coating of Yeast Cells with Polyelectrolytes and MWNTs. In this study, S. cerevisiae cells were layer-by-layer coated with polyelectrolytes and MWNTs using the alternating deposition of PAH and PSS on the surface of the cells. Since the bare yeast cells exhibit a low negative charge, the positively charged PAH monolayer was first deposited on the surface of the yeast cells by dropwise introduction of 300 μL of the yeast cell aqueous suspension (10 mg mL-1) into 1 mL of PAH (1 mg mL-1 in 0.5 M NaCl) during constant vortexing and incubated while shaking for 15 min. Then, the cells were separated by centrifugation, the remaining excess of the polyelectrolyte was discarded, and the cells were washed thrice with water. Then, the cells were similarly introduced into the PSS (1 mg mL-1 in 0.5 M NaCl) solution, followed by incubation for 15 min while shaking, centrifugation, and washing. Next, the third PAH monolayer was deposited as described above. After washing with water, the PAH/ PSS/PAH coated cells were introduced into the aqueous suspension of negatively charged (due to carboxyl and hydroxyl groups introduced by oxidation treatment) MWNTs (1 mg mL-1) and incubated for 30 min while shaking, followed by centrifugation and washing with water in order to remove any unattached MWNTs. Finally, in order to seal the MWNTs on the surface of the cells the two additional polyelectrolyte layers (PAH/PSS) were similarly deposited onto the cells, and the resulting coating architecture was PAH/PSS/PAH/MWNTs/PAH/PSS. In several experiments, FITC-PAH (0.5 mg mL-1 in 0.5 M NaCl) was used instead of PAH to examine the integrity of polyelectrolyte layers with fluorescence microscopy. In this case, the resulting coating architecture was PAH/PSS/PAH/MWNTs/FITC-PAH/PSS. Viability Test. The viability of the native and polyelectrolyte/ MWNT-coated yeast cells was tested by examining the activity of intracellular esterases and membranes integrity using FDA.31 Since FDA is poorly soluble in water, it was first dissolved in acetone at 10 mg mL-1 (stock solution), and then an aliquot (10 μL) of the stock solution was mixed with 490 μL of water and the appropriate volume of the aqueous FDA was added into the suspension of the yeast cells until the final concentration of 0.02 mg mL-1 (pH ∼ 6.5) was reached. The suspension was incubated for 20 min at room temperature while shaking, and then the cells were collected by centrifugation, washed with water, and examined using a fluorescent microscope (as described below). Optical and Fluorescence Microscopy. Bright field optical images of the bare and polyelectrolyte/MWNT-coated cells were obtained using a Leica DM 1000 (Germany) upright microscope equipped with an immersion oil 100 objective (numerical aperture 1.25) and a Leica DFC 290 CCD digital camera. Optical images of the bare and polyelectrolyte/MWNT-coated cells were obtained at the identical illumination conditions and CCD-camera signal acquisition settings. Fluorescence microscopy images were obtained using a Leica DMIL (Germany) inverted microscope equipped with a 40 objective (numerical aperture 0.5) and with a Leica DFC 420 CCD digital camera using (31) Breeuwer, P.; Drocourt, J.-L.; Bunschoten, N.; Zwietering, M. C.; Rombouts, F. M.; Abee, T. Appl. Environ. Microbiol. 1995, 61, 1614–1619.

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Article the green excitation filter (488 nm) and a mercury lamp. Samples were imaged immediately after preparation. Transmission Electron Microscopy. Transmission electron microscopy (TEM) images of thin slices of native and polyelectrolyte/MWNT-coated yeast cells were obtained using a Jeol 1200 EX microscope (Japan) operating at 80 kV. Cells were fixed using 2.5% glutaraldehyde and 1.5% paraformaldehyde in phosphate buffer (0.1 M at pH 7.4) for 2 h at room temperature and postfixed in 1% OsO4 for 1 h. After dehydration, the cells were embedded in Epon resin (PELCO Eponate 12 Kit); then, the ultrathin sections were cut with a diamond knife using an Ultracut III ultramicrotome (LKB, Sweden) and mounted on Formvarcoated 100-mesh copper grids. Sections were stained with 2% aqueous uranyl acetate and with lead citrate at room temperature. Scanning Electron Microscopy. Scanning electron microscopy (SEM) images were obtained using a Carl Zeiss Evo-40 SEM (Germany) operating at 10 kV. Samples of native and polyelectrolyte/MWNT-coated yeast cells were prepared by dispersing 5 μL of a cellular suspension on the surface of acetone washed specimen stubs and dried at þ37 °C overnight. Then, the cells were sputter-coated with a 5-nm-thick gold layer using a Baltec SCD 005 sputter-coater.

Electrochemical Characterization of Polyelectrolyte/ MWNT-Coated Yeast Cells. Cyclic voltammetry was investigated using a voltammetric analyzer Ecotest-VA (Econix-Expert, RF). Electrochemical impedance spectroscopy (EIS) measurements were performed using Autolab PGSTAT 302 instrument (Netherlands) equipped with an FRA module for impedance measurement and NOVA 1.4 software. Six different samples covered with polyelectrolytes and bare or modified yeast cells (as described below) were used for electrochemical measurements.

Immobilization of Yeast Cells onto Glassy Carbon Electrodes. Glassy carbon electrodes (2.5 cm rod, L 2.2 mm, pressed in a polytetrafluoroethylene tube) were mechanically cleaned with alumina and rinsed sequentially with acetone, NaOH, and H2SO4. Next, the electrodes were immersed into 0.2 M H2SO4 and 10 potential cycles were run in the range from -400 to 800 mV vs Ag/AgCl reference electrode at a scan rate of 250 mV s-1 to remove electrochemically active impurities. After drying, 2 μL of the polyelectrolytes (1 mg mL-1 PAH or PSS in 0.5 M NaCl) were sequentially deposited onto the electrodes starting from the PAH layer, followed by washing with water after each deposition step, until the coating architecture was PAH/PSS/PAH/PSS/PAH. Then, the yeast cells were introduced on the surface of the electrode (10 mg mL-1). The following cell specimens were used in this study: bare living yeast cells, polyelectrolytes/MWNTs-coated living yeast cells, bare thermally inactivated yeast cells, and polyelectrolytes/MWNTs-coated thermally inactivated yeast cells. Finally, two additional polyelectrolyte bilayers (PAH/PSS/PAH/PSS) were placed above the immobilized cells; the electrodes were washed and used for voltammetry and EIS measurements. The resulting surface architecture was PAH/PSS/PAH/PSS/PAH/cells/PAH/PSS/PAH/ PSS. In addition, control experiments with the electrodes modified with (PAH/PSS)4 layers only were performed. Voltammetry Experiments. Cyclic voltammograms were recorded using 1 mM aqueous K3[FeCN]6 working solution at the scan rate 50 mV s-1. Potential runs were repeated five times with intermediate stirring for 3 min. For the washing step experiments, the electrode was transferred into 0.5 M NaCl solution and the voltammograms were recorded again as described above at the scan rate of 50 mV s-1. EIS Measurements. The impedance data were recorded in 0.5 M NaCl containing 0.01 M K3[Fe(CN)6] and 0.01 M K4[Fe(CN)6] at 0.315 V (formal potential of [Fe(CN)6]3-/4-) within the frequency range from 100 kHz to 0.04 Hz with the alternate amplitude voltage 5 mV. Calculations were performed using FRA module for impedance measurement and NOVA 1.4 software and R(C1, R1)(C2, R2) equivalent circuit. DOI: 10.1021/la902937s

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Figure 1. Schematic representation of the polyelectrolyte-mediated assembly of multiwalled carbon nanotubes on living yeast cells.

Results and Discussion LbL Deposition of Polyelectrolytes and MWNTs. The scheme of our method is sketched in Figure 1 and consists of the following stages: (i) individual living cells are coated by the consecutive deposition of polyelectrolytes (1 mg mL-1 in 0.5 M NaCl) starting with PAH, until the desired coating architecture (PAH/PSS/PAH) is obtained; (ii) polyelectrolyte-modified yeast cells modified with an outer positively charged PAH layer are introduced into the aqueous dispersion of oxidized negatively charged MWNTs (1 mg mL-1); (iii) another polyelectrolyte bilayer (PAH/PSS) is deposited onto the polyelectrolyteMWNTs-modified yeast cells to prevent the removal of the immobilized MWNTs from the cell walls. The strategy adopted for this study is based on the previous papers reporting the coating of mammal and microbial cells with polyelectrolyte layers alone4,32 or with polyelectrolyte layers doped with spherical metal or silica nanoparticles with diameters from 20 to 80 nm.5,9,10 Here, we extend this technique in order to apply the LbL assembly of polyelectrolytes to coat yeast cells with rod-shaped carbon nanotubes. We used commercially available synthetic polyelectrolytes PAH and PSS and MWNTs with outer diameter around 10-30 nm and length distribution from 0.5 to 500 μm (as provided by the supplier; the representative SEM image of MWNTs used in this study is shown in Figure S1, Supporting Information). We found that the coating of native yeast cells with PAH/PSS and MWNTs leads to the effective changes in color of the cells, which can be monitored using optical microscopy. The typical optical microscopy images of the native and the polyelectrolytes/ MWNTs-coated cells are given in Figure 2. As one can see, the polyelectrolyte-mediated deposition of MWNTs onto the surfaces of yeast cells strongly affects the color of the yeast cells which appear to be dark green after the deposition of MWNTs; in addition, several aggregates of the nanotubes can be distinguished on the optical microscopy images (it should be mentioned that the images shown in Figure 2a,b were taken almost simultaneously under the same illumination conditions and CCD camera acquisition settings and the sample was placed on a single glass slide; therefore, the difference in appearance of the cells is due to polyelectrolyte-mediated MWNTs coating only). This tremendous change in color of the MWNT-modified yeast cells indicates that the cells are coated with a dense layer of carbon nanotubes; this assumption will be further confirmed using TEM and SEM. Along with nonfluorescent PAH, in a set of experiments we used FITC-labeled PAH (FITC-PAH) to produce an outermost bilayer (FITC-PAH/PSS) on the surface of polyelectrolyte/ MWNT-coated cells, which allowed us to demonstrate the integrity and uniformity of the polyelectrolyte coatings. The (32) Franz, B.; Balkundi, S. S.; Dahl, C.; Lvov, Y. M.; Prange A. Macromol. Biosci. 2009, 9; DOI: 10.1002/mabi.200900142

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Figure 2. (a) optical microscopy image of the native yeast cells; (b) optical microscopy image of the yeast cells coated with PAH/ PSS/PAH/MWNTs/PAH/PSS; (c) fluorescence microscopy image of the yeast cells coated with PAH/PSS/PAH/MWNTs/FITCPAH/PSS.

representative fluorescence microscopy image of yeast cells coated with polyelectrolyte shells incorporating MWNTs (Figure 2c) shows that the cells are coated with the fluorescent FITC-PAH adsorbed onto the MWNT/polyelectrolyte-coated cells. The variations of fluorescence intensity across individual cells is likely to originate from the diffusion of FITC-PAH in the cell walls and perhaps into the cytoplasm, as was shown in a recent report presenting the polyelectrolyte nanoassembly of polyelectrolytes on various bacteria.32 As expected, no dark areas originating from MWNT aggregates were observed because the FITC-PAH layer is deposited above MWNTs immobilized on the cell walls using the polyelectrolyte-mediating technique. Langmuir 2010, 26(4), 2671–2679

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Figure 3. (a) TEM image of a native yeast cell; (b) TEM image of a yeast cell coated with PAH/PSS/PAH/MWNTs/PAH/PSS; (c) a highmagnification TEM image of the cell wall of an yeast cell coated with PAH/PSS/PAH/MWNTs/PAH/PSS. Black arrows indicate the MWNTs aggregates, white arrows the individual nanotubes.

We applied transmission electron microscopy to further investigate the thin structure of the polyelectrolyte/MWNT-modified cells (while the native cells were used as the control samples). TEM images of the resin-embedded thin slices of the native and polyelectrolyte/MWNT-coated yeast cells are presented in Figure 3. As one can see, originally smooth and featureless cell wall surface of the native yeast cells (Figure 3a) after the deposition of polyelectrolytes and MWNTs appears to be “hairy” due to the solution-oriented partially immobilized long (up to several hundred nanometres) nanotubes (Figure 3b,c). TEM images indicate that a continuous layer of MWNTs is produced as a result of the sequential polyelectrolyte/carbon nanotube deposition cycles. The thickness of the MWNTs layer, as measured from TEM images, is around 80 nm, although several areas of the cell walls coated with considerable thinner (20-30 nm) and thicker layers (up to 200 nm) can be found occasionally. It was shown previously that the thickness of polyelectrolyte films can be estimated using quartz crystal microbalance (QCM) techniques, and the results showed that the thickness of PAH/PSS layer is around 1 nm.5 QCM measurements utilizing the resonant frequency shifts caused by the surface mass increases are regarded as a versatile technique for the thickness determination of uniform thin films; however, QCM data obtained using optically polished metal-coated quartz resonators as the starting surface for the film growth do not necessarily represent the thickness of the films assembled on the rough and nonuniform cell walls. Moreover, as was demonstrated recently, the thickness of carbon nanotubes and polyelectrolyte assemblies measured with QCM is considerably smaller than if measured using other techniques (UV-vis spectroscopy).20 Therefore, we resorted to using TEM images of thin sections of the MWNT/polyelectrolyte-coated cells to estimate the overall thickness of the composite nanolayers. Since the thickness of the polyelectrolyte layers alone is negligible in comparison with the MWNTs layer, the overall thickness of the coating is determined by the MWNTs deposited on the adjacent polyelectrolyte layer. TEM images indicate that the MWNTs are either deposited as a monolayer when a single nanotube is immobilized on the cell wall or form aggregated bundles. These aggregates may originate from the originally aggregated nanotubes or can be spontaneously formed after the deposition; Langmuir 2010, 26(4), 2671–2679

supposedly, both mechanisms can exist simultaneously. As noticed above, the nanotubes are oriented randomly; some of them are completely bound to the cell wall interface, while the remaining are exposed outside the cell walls (determining the “hairy” appearance of the encapsulated cells). This observation suggests that the polyelectrolyte multilayers provide the effective deposition and “sealing” of MWNTs on the cell walls. Due to the aggregation of the nanotubes, a number of pores is clearly seen in the MNWTs layer. Presumably, the porous character of the MWNTs layer may retain the electric and adsorptive properties of pristine MWNTs, thus making it possible to use MWNTs assembled on the yeast cells in bioelectronic applications. We suppose that the monolayer of MWNTs does not prevent the transport of low-molecular-weight molecules (which will be demonstrated later). We also studied the structure of the MWNT/polyelectrolyte coatings deposited on the cell walls of yeast cells by SEM. The representative images of the native and polyelectrolyte/MNWTcoated yeast cells are given in Figure 4. As seen on the images, the yeast cell walls are coated with a fairly uniform MWNT/ polyelectrolyte composite layer; therefore, the appearance of the coated cells contrasts considerably with the native cells. It is noteworthy that several MWNTs “tails” are clearly visible on the cells, which are apparently produced by the individual nanotubes or very thin nanotube aggregates (similar to those seen in Figure 3c). These unbound MWNT residues can be potentially utilized in microbridging and other similar applications. Microscopy studies demonstrate that the LbL approach to fabricate polyelectrolyte multilayers doped with inorganic MWNTs can be regarded as the effective and versatile way to create hybrid cell-based microparticles. After the inspection of the multiple fields during TEM and SEM imaging, we conclude that the majority of the cells are uniformly coated with MWNTs, whereas some are apparently partially coated, which might result from the mechanical impact during the sample preparation. As it was shown earlier, the LbL approach is applicable for immobilization of noble metal9 and silica8,10 nanoparticles on the surfaces of yeast cells and bacterial spores, and here, we extend this technique for the deposition of wire-like nanostructures on the cell walls. We suppose that this technique can be used for the DOI: 10.1021/la902937s

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Figure 4. (a) SEM image of a native yeast cell; (b) SEM image of yeast cells coated with PAH/PSS/PAH/MWNTs/PAH/PSS; (c) a highmagnification SEM image of a single yeast cell coated with PAH/PSS/PAH/MWNTs/PAH/PSS.

Figure 5. Viability test of the polyelectrolyte/MWNT-coated yeast cells after the incubation in 0.02 mg mL-1 aqueous FDA: (a) bright field microscopy image; (b) corresponding fluorescence microscopy image; (c) merged bright field and fluorescence microscopy image.

immobilization of other types of nanotubes and nanorods (i.e., single-walled carbon nanotubes, peptide nanotubes, cellulose nanowhiskers, magnetic nanorods, etc). In addition, on the basis of our previous results,9 we believe that other polyelectrolytes (including biomacromolecules) might be used in order to facilitate the immobilization of MWNTs. Taking into account the average intracellular volume (70 μm3) and the membrane surface (85 μm2) of yeast cells,31 we suppose that the yeast cells might be utilized as convenient templates for the assembly of carbon nanotubes to produce well-defined core-shell microparticles. Since the cellular cores can be easily removed by either calcination33 or enzymatic treatment,34 the hollow MWNTs microcapsules may potentially be obtained via the technique described here. This research is being currently performed by our group and will be reported later. Viability Study of Polyelectrolyte/MWNT-Coated Yeast Cells. The utilization of nanomaterials, especially single-walled and multiwalled carbon nanotubes in biological applications and devices, often raises serious cytotoxicity and genotoxicity concerns.35,36 Diaspro and co-workers reported earlier that the viability of the yeast cells coated by up to six PAH/PSS bilayers is not affected; moreover, the budding of the polyelectrolytecoated yeast cells was demonstrated.4 Even the polyelectrolyte shells doped with noble metal nanoparticles9 do not jeopardize the viability of the encapsulated fungi cells. Therefore, we assumed that the viability in our case should not be affected by polyelectrolyte layers alone. In our study, we demonstrate that (33) Baia, B.; Wang, P.; Wu, L.; Yang, Li.; Chen, Z. Mater. Chem. Phys. 2009, 114, 26–29. (34) Salazar, O.; Asenjo, J. A. Biotechnol. Lett. 2007, 29, 985–994. (35) Isobe, H.; Tanaka, T.; Maeda, R.; Noiri, E.; Solin, N.; Yudasaka, M.; Iijima, S.; Nakamura, E. Angew. Chem., Int. Ed. 2006, 45, 6676–6680. (36) Zhu, L.; Chang, D. W.; Dai, L.; Hong, Y. Nano Lett. 2007, 7, 3592–3597.

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MWNTs are successfully immobilized on the surface of the yeast cells via the PAH/PSS thin layers; therefore, it was important to investigate whether this type of coating is cytotoxic. The viability of the yeast cells coated with PAH/PSS/PAH/MWNTs/PAH/ PSS was studied using the FDA test, which is routinely used to estimate the viability of eukaryotic cells.31 The FDA-mediated approach is based on the esterase activity of the living cells, where the originally nonfluorescent FDA molecules travel through the cell walls and undergo esterase-catalyzed hydrolysis inside the cells, resulting in the formation of a fluorescent product, which indicates that the intracellular enzymes and cell membranes are intact. The results of the viability test are presented in Figure 5, where the bright field microscopy images, corresponding fluorescent images, and the merged bright field and fluorescence microscopy images of polyelectrolyte/MWNT-coated cells incubated in aqueous FDA solution and then washed with water are presented. In Figure 5, the green FDA-mediated fluorescence indicates that the yeast cells preserve their viability after coating with polyelectrolytes/MWNTs. The experiments were performed at pH ∼ 6.5, which is close to the optimal pH for the FDA enzymatic hydrolysis by yeast cells esterases.31 Dark spots clearly seen on the several cells are caused by the large aggregates of MWNTs, which reduce the fluorescence intensity, although on the merged (bright field and fluorescence) image the green fluorescence is still visible; therefore, the viability of the cells is not affected by the deposition of MWNTs. As was suggested in earlier reports, the polyelectrolyte multilayers may act as protective barriers preventing the intrusion of nanomaterials into the cells.4,5,9 Our data suggest that the polyelectrolyte/MWNT composite layers do not inhibit the uptake of the low-molecular-weight molecules (such as FDA); therefore, we believe that these coatings are apparently permeable Langmuir 2010, 26(4), 2671–2679

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for other low-molecular-weight molecules (including nutrients). Therefore, we suppose that the approach described in the present paper is more applicable if the living yeast cells are required. Alternatively, we used thermally inactivated (by boiling for 2 h) yeast cells as the templates for the polyelectrolyte-mediated MWNTs deposition; in this case, all the cells were dead after the inactivation (optical and fluorescence images of the thermally inactivated yeast cells are shown in Figure S2, Supporting Information). The thermal treatment of yeast cells by boiling for several hours is a well-established technique37 for relatively gentle inactivation of the cells due to the cellular proteins denaturation resulting after the prolonged heating. We suppose that the approach described here can be applied for the development of microdevices (i.e., microbridges) where the cell viability can be compromised (by using the already thermally inactivated or chemically fixed cells), i.e., all the potential applications which require the cells as well-defined and highly accessible templates regardless of the biological functionalities of the cells. Particularly, the approach to modify cells with carbon nanotubes can be potentially expanded to other species, including bacteria and algae, possessing a number of peculiar shapes, therefore finding applications for the fabrication of cell-based conductive coatings similar to those described by Agarwal et al. using wood microfibers.20 Obviously, the additional studies are required to investigate the long-term viability effects of the polyelectrolyte/ MWNT coatings onto yeast cells. However, we believe that, as long as the approach proposed here is apparently non-cytotoxic and the coated cells are metabolically active, we suppose that the technique described here can be used in the development of hybrid bioelectronic devices (i.e., cell-based biosensors38 or microfluidic devices39) that would benefit from the biological activity of the cells and electrochemical properties of carbon nanotubes. Electrochemical Characterization of Polyelectrolyte/ MWNT-Coated Yeast Cells. Here, we demonstrate that this technique can be used in the electrochemistry studies of the electrode-immobilized living and dead native and polyelectrolyte/MWNT-coated yeast cells. For this purpose, the electron transfer was recorded using ferricyanide as an artificial electron acceptor. A similar approach was successfully used for the fast and reliable detection of biochemical oxygen demand (BOD),38,40,41 ethanol,38 formaldehyde,42 and toxicity screening.43 In the latter case, the relative changes in the current of [Fe(CN)6]3- reduction were measured after incubating Escherichia coli living cells in the presence of heavy metals and other antimicrobial agents. [Fe(CN)6]3- system is commonly used in microbial biosensors developed for BOD measurements. Typically, microbial BOD sensors demonstrated fairly good characteristics of stability and long-term lifetime.41,44 The behavior of ferricyanide-based systems does not differ dramatically from the other artificial mediators (i.e., 2,6-dichlorophenolindophenol). The regeneration of the mediator is performed during the electrochemical reaction. The direct current measurements are (37) Autio, K.; Matilla-Sandholm, T. Appl. Environ. Microbiol. 1992, 58, 2153– 2157. (38) Baronian, K. H. R. Biosens. Bioelectron. 2004, 19, 953–962. (39) Garcı´ a-Alonso, J.; Greenway, G.; Hardege, J.; Haswell, S. Biosens. Bioelectron. 2009, 24, 1508–1511. (40) Catterall, K.; Morris, K.; Gladman, C.; Zhao, H.; Pasco, N.; John, R. Talanta 2001, 55, 1187–1194. (41) Chen, H.; Ye, T.; Qiu, B.; Chen, G.; Chen, X. Anal. Chim. Acta 2008, 612, 75–82. (42) Khlupova, M.; Kuznetsov, B.; Demkiv, O.; Gonchar, M.; Cs€oregi, E.; Shleev, S. Talanta 2007, 71, 934–940. (43) Liu, C.; Sun, T.; Xu, X.; Dong, S. Anal. Chim. Acta 2009, 641, 59–63. (44) Morris, K.; Catterall, K.; Zhao, H.; Pasco, N.; John, R. Anal. Chim. Acta 2001, 442, 129–139.

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performed in the cyclic voltammetry mode to ensure that the full cycle of oxidation/reduction is performed in each measurement. This provides the regeneration cycle for [Fe(CN)6]3- and hence the recovery of bioelectrochemical system. The peak current values and their ratio indicate the efficiency of electron transfer in the microbiological path of the cycle. Therefore, here we adapted the method described in ref 42 to immobilize the native and polyelectrolyte/MWNT-coated yeast cells on the glassy carbon electrodes. In this paper, we used living and dead cells (both bare and polyelectrolyte/MWNT-coated) which were incorporated between polyelectrolyte layers deposited on the electrode surfaces, and the voltammetric and impedimetric responses were recorded using [Fe(CN)6]3- redox probe. The schematic representation of the modification of the glassy carbon electrodes with polyelectrolytes and yeast cells (both native and polyelectrolyte/MWNT-coated) is given in Figure 6a. Since the polyelectrolyte/MWNT-coated yeast cells are viable, we applied lowmagnification epifluorescence microscopy to demonstrate the living cell layer at the top of the electrode (Figure 6b; note the green fluorescence emitted by the living yeast cells). Voltammetry Studies. At the first stages, we investigated the voltammetry of the polyelectrolyte multilayer coating alone, with no cells embedded between the polymer layers. The typical cyclic voltammograms are given in Figure S3, Supporting Information. We found that the behavior of ferricyanide anion on the glassy carbon electrode covered with (PAH/PSS)4 was quasi-reversible with nearly equal anodic and cathodic peaks and the formal redox potential (E0 = þ200 mV vs Ag/AgCl) shifted to a less positive value against that on the bare electrode (þ295 mV). This can be related to the moderated electron transfer through the nonconductive barrier of (PAH/PSS)4 coating. Linear dependence of the peak currents on the square root of the scan rate (not shown) indicates the diffusional control of the electrode reaction. In repeated measurements from the same solution, the reduction peak current increased significantly after the first scan and then remained unchanged for more than 30 min. We demonstrate that the incorporation of the living cells in the polyelectrolyte layer resulted in the sufficient decrease of the [Fe(CN)6]3- reduction current probably because of its participation in the oxidative electron transfer chain of living yeast cells. This was confirmed by similar experiments with the thermally inactivated yeast cells, which showed much lower efficiency of this process and hence increasing current level as shown in Figure 6c. The difference in the reduction peak current for living and dead cells was found to be 2.2 ( 0.1 μA, or about 20% of the initial current observed for PAH/PSS multilayers in the absence of yeast cells. The cyclic voltammograms of [Fe(CN)6]3- obtained with the yeast cells encapsulated with polyelectrolyte/MWNT were similar to those of PAH/PSS covered electrodes with a wellpronounced oxidation/reduction coupled peak indicating the reversibility of the electron transfer. The [Fe(CN)6]3- reduction current decreased to 9.0 ( 0.2 μA for polyelectrolyte/MWNTencapsulated living cells immobilized on the glassy carbon electrode and to 12.0 ( 0.1 μA for polyelectrolyte/MWNT-encapsulated dead cells. Thus, the difference observed for living and thermally inactivated cells coated with polyelectrolytes and MWNTs was higher than that observed for the bare cells. Meanwhile, the efficiency of electron transfer observed for living cells and quantified as the decay of the reduction current against (PAH/PSS)4 layer deposited onto the glassy carbon electrode was found to be higher for polyelectrolyte/MWNT-coated living yeast cells, which is probably due to the partial electrostatic repulsion of negatively charged redox probe anion from the MWNTs coating. The difference in the behavior of living and dead cells in the DOI: 10.1021/la902937s

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Figure 6. (a) Schematic representation of the immobilization of yeast cells onto glassy carbon electrodes. (b) Low-magnification optical microscopy image of the glassy carbon electrodes coated with polyelectrolyte/MWNT-coated yeast cells (the inset shows the green FDAmediated fluorescence in the layer of the living cells). (c) Changes in the peak currents of [Fe(CN)6]3- reduction during the incubation of the electrode covered with PAH/PSS/MWNTs/living cells and PAH/PSS/MWNTs/dead cells in 1.0 mM [Fe(CN)6]3- solution (at the scan rate 50 mV/s) and during the washing step.

polyelectrolyte layers was also observed during the washing step. The electrodes were first incubated in the [Fe(CN)6]3- probe solution and then moved to 0.5 M NaCl solution that contained no indicator. Typically, the currents related to [Fe(CN)6]3reduction decayed within 10 min indicating fast release of the redox probe from the surface layer. However, for the electrode covered with polyelectrolyte/MWNT-coated living cells, the reduction current first increased by about 15% of initial value so that the difference in the signals related to living and dead cells within the 3 min washing step reached about 50% of the initial value. This is remarkably sufficient to distinguish living and inactivated cells embedded in the electrode surface layer. These results demonstrate that the interaction between polyelectrolyte/ MWNT-coated living cells and [Fe(CN)6]3- probe was much stronger than that for bare living cells. We suppose that this phenomenon is likely to be used in respiratory microbial sensors or in antimicrobial tests in order to increase their sensitivity in viability testing of living cells. EIS Measurements. The difference in the electron transfer ability and mediation efficiency was also studied using the EIS experiments with the equivalent mixture of [Fe(CN)6]3-/4- as a redox probe. The R(C1, R1)(C2, R2) equivalent circuit previously described for charged nonconductive DNA layers deposited on polyelectrolyte support was used for the calculations.45 On the Nyquist diagram (shown in Figure S4, Supporting Information), two semicircles corresponding to the equivalent circuits (R1, C1) at high frequencies and (R2, C2) at low frequencies were defined. Although the behavior of capacitance at low frequencies differs (45) Gautier, C.; Cougnon, C.; Pilard, J.-F.; Casse, N. J. Electroanal. Chem. 2006, 587, 276–283.

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Table 1. Electrochemical Impedance Characteristics of Glassy Carbon Electrodes Coated with Polyelectrolyte Layers Incorporating Bare and Polyelectrolyte/MWNT-Coated Yeast Cells R1, Ω C1, μF R2, kΩ C2, μF

modifying layer (PAH/PSS)4 bare living cells bare dead cells PAH/PSS/MWNTs-coated living cells PAH/PSS/MWNTs-coated dead cells

360 232 131 180 176

0.085 0.244 0.646 0.320 0.352

5.15 5.62 6.50 5.57 4.59

76.07 37.2 92.9 72.7 68.2

from the ideal, the coefficient R of the expression of the impedance Z(ω) = Const(jω)-R was calculated in the range 0.89-0.95 (where ω is angular frequency). This proved that C2 showed near-capacitance behavior in the second circuit. First and second circuits can be attributed to the charge transfer resistance and capacitance of the electrode/surface layer and surface layer/ solution interfaces, respectively, and R to the sum of the electrolytic resistance and that of the electrode material. The R value calculated during these experiments was constant and equal to ∼110 Ω. Other results of impedimetric measurements are summarized in the Table 1. All the measurements were performed in six replicas and the standard deviation of appropriate C and R values did not exceed 5%. In compliance with the voltammetric data, the efficiency of the charge transfer on the internal interface (electrode/polyelectrolyte composite) substantially increased in the case of yeast cells embedded into the polyelectrolyte layers on the electrode surface. This effect is more pronounced for living cells. The capacitance generally changes in the direction opposite that of charge transfer resistance. This can be explained by the charge separation due to the transport of the anionic redox probe through the Langmuir 2010, 26(4), 2671–2679

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LbL-deposited polyelectrolyte layers. On the external interface surface (layer/solution interface), the resistance shifts coincide well with appropriate values of reduction currents and hence can also be attributed to the effect of the yeast cells and their microenvironment on the redox conversion of [Fe(CN)6]3-/4ions. As in the case of voltammetric experiments, the use of polyelectrolyte/MWNT coatings deposited on the yeast cells increases both the signal difference between living and inactivated cells and the accuracy of measurement in comparison with the bare cells implemented in polyelectrolyte layers.

Conclusions To conclude, we present a simple and versatile technique for the directed polyelectrolyte-mediated assembly of MWNTs on the microbial cells. We demonstrate that the layer-by-layer deposition of PAH/PSS and MWNTs allows for the effective immobilization of MWNTs on the surfaces of yeast cells. As indicated by the enzyme activity viability test using FDA, the cells encapsulated into polyelectrolyte/MWNT composite coating preserve their viability. Comparative investigation of the bare and polyelectrolyte/ MWNT-coated yeast cells (both living and thermally inactivated) implemented in the polyelectrolyte layers of PAH/PSS on the glassy carbon electrodes shows that the use of MWNTs increased both the difference in the charge transfer and the sensitivity of the

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response especially in the case of washing the electrodes after their incubation in the redox probe. The possibility of distinguishing living and inactivated cells is important for creating fast and inexpensive microbial sensors for the detection of toxic features of waters or testing antimicrobial activity of disinfectants. We believe that the technique presented here may find a wide range of applications in bioelectronics and the development of novel materials. Acknowledgment. We thank Dr. V. N. Paunov, Dr. A. P. Kiyasov, and Dr. M. Culha for their support, Mr. M. Kahraman and Mr. Y. Osin for SEM images, Dr. V. V. Salnikov for TEM images, and anonymous reviewers for their valuable suggestions. We appreciate the financial support by the Academy of Sciences of the Republic of Tatarstan (grant 14-15/(G)-2009). The study is dedicated to the loving memory of the late Prof. V. G. Vinter (1939-2005). Supporting Information Available: Additional figures demonstrating the SEM images of MWNTs used in this study; optical and fluorescence images of the thermally inactivated yeast cells; cyclic voltammograms; Nyquist diagram scheme. This material is available free of charge via the Internet at http://pubs.acs.org.

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