pubs.acs.org/Langmuir © 2009 American Chemical Society
Living Fungi Cells Encapsulated in Polyelectrolyte Shells Doped with Metal Nanoparticles Rawil F. Fakhrullin,*,† Alsu I. Zamaleeva,† Mikhail V. Morozov,† Diana I. Tazetdinova,† Farida K. Alimova,† Albert K. Hilmutdinov,† Renat I. Zhdanov,‡ Mehmet Kahraman,‡ and Mustafa Culha*,‡ †
Department of Biochemistry, Kazan State University, Kremlevskaya 18, Kazan 420008, Tatarstan, Russian :: Federation and ‡Department of Genetics and Bioengineering, Yeditepe University, Kayıs-da gı/Kadıkoy, Istanbul 34755, Turkey Received November 22, 2008. Revised Manuscript Received January 29, 2009
We report the layer-by-layer coating of living fungi cells (Saccharomyces cerevisiae and Trichoderma asperellum) with polyelectrolytes poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) and bovine serum albumin/DNA and citrate-stabilized gold and silver nanoparticles. It was found that the nanoparticles were effectively incorporated between oppositely charged polyelectrolyte layers, modifying the topography and the roughness of cell walls. The formation of large aggregates of nanoparticles on the cell walls of encapsulated cells was shown. It was found that the encapsulated cells preserved their viability and the shells were soft enough to allow the growth of mycelium. The surface-enhanced Raman scattering (SERS) was used to investigate the biochemical environments of the gold and silver nanoparticles immobilized on the surface of T. asperellum conidia. The SERS spectra from encapsulated conidia and polyelectrolytes indicate that both gold and silver nanoparticles interact with cell walls from different locations, and nanoparticle-polyelectrolyte interaction is limited. The approach described in this paper might have potential applications in modification of living cells.
Introduction Microorganisms, including microscopic fungi, are widely used in a variety of medical, biotechnological, and industrial applications. A great deal of effort is focused worldwide to increase the efficacy of microorganism-based systems and devices via various modification and immobilization techniques. Some very important directions in current research can be pointed out, namely, the development of novel experimental approaches for the effective immobilization of microorganisms on various surfaces1-3 and selective detection of minute amounts of microbial cells.4-7 Nanoparticles are regarded as promising tools for cellular modification, especially in the development of microdevices; therefore, a number of recent reports focus on the modification of cells with nanoparticles. Berry et al. deposited a monolayer of gold nanoparticles on the bacteria (E. coli) surface using specific peptide-bacteria affinity in order to produce electrically conducting microbridges.8 Another approach of the modification was described by Jing et al.9 Electroless chemical plating process was applied to fabricate nickel-coated bacterial cells (E. coli), and it was revealed that after metallization a number of nickel nanoparticles were *Corresponding authors. E-mail:
[email protected] (M.C.).
[email protected] (R.F.F.);
(1) Krol, S.; Nolte, M.; Diaspro, A.; Mazza, D.; Magrassi, R.; Gliozzi, A.; Fery, A. Langmuir 2005, 21, 705. (2) Kubota, M.; Matsui, M.; Chiku, H.; Kasashima, N.; Shimojoh, M.; Sakaguchi, K. Appl. Environ. Microbiol. 2005, 71, 8895. (3) Suo, Z.; Avci, R.; Yang, X.; Pascual, D. W. Langmuir 2008, 24, 4161. (4) Jarvis, R. M.; Goodacre, R. Anal. Chem. 2004, 76, 40. (5) Jarvis, R. M.; Brooker, A.; Goodacre, R. Anal. Chem. 2004, 76, 5198. (6) Premasiri, W. R.; Moir, D. T.; Klempner, M. S.; Krieger, N.; Jones, G. I.; Ziegler, L. D. J. Phys. Chem. B 2005, 109, 312. (7) Laucks, M. L.; Sengupta, A.; Junge, K.; Davis, E. J.; Swanson, B. D. Appl. Spectrosc. 2005, 59, 1222. (8) Berry, V.; Rangaswamy, S.; Saraf, R. F. Nano Lett. 2004, 4, 939. (9) Wang, J.; He, S. Y.; Xu, L. N.; Gu, N. Chin. Sci. Bull. 2007, 52, 21.
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observed to be equally distributed at the bacterial surface with high uniformity. The viability of cells was not investigated, though. In situ reduction of Au3+ ions by bacterial cells resulting in the formation of gold nanoshells around E. coli cells has been recently reported.10 The mechanisms of metal ion reduction by bacterial extracellular structure are still not clear; the procedure of coating requires the incubation of cells in Au3+ aqueous solutions for 32 h, which leads to formation of 7-8 nm thick nanoparticles, while the bacterial cells remain alive. Safarik et al. reported the modification of yeast cells with magnetic nanoparticles in order to prepare a magnetically responsive biocomposite material; nanoparticles were directly adsorbed onto the surfaces of cells by mixing the cellular suspension and as-synthesized ferrofluid.11 Microscopic fungi coated with nanoparticles are regarded as suitable living templates for the development of microwires. Recent reports describe the utilization of the following strategy: first, the nanoparticles are modified somehow in order to increase their affinity to the fungal cell wall; next, the spores are introduced into the media containing thus-modified nanoparticles and allowed to germinate. Hence, the nanoparticles can be assembled on the surface of the growing mycelium. Li and coauthors reported the coating of living hyphae of Aspergillus niger fungus with oligonucleotide-modified gold nanoparticles which formed a dense layer on the mycelium. The mechanism of interaction of DNA-modified nanoparticles with maturing mycelium is not clear yet, although the technique described allows additional manipulations with the mycelium to be performed, such as the introduction of secondary complementary oligonucleotide-modified nanoparticles and, (10) 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, 4430. (11) Safarik, I.; Rego, L. F. T.; Borovska, M.; Mosiniewicz-Szablewska, E.; Weyda, F.; Safarikova, M. Enzyme Microb. Technol. 2007, 40, 1551.
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as a result, the formation of double-layered nanoparticle ring structures.12 Recently, Sugunan et al. applied the similar route to coat several filamentous fungi with glutamate-stabilized gold nanoparticles, which provided the maturing hyphae with the only nutrition source available in the growth media.13 As a result, the formation of a dense and apparently multilayered coating of nanoparticles was documented. Both reports postulate the usefulness of such structures in the development of a range of novel biomimetic materials. Obviously, the direct placement of nanoparticles onto living cells can severely affect the viability of cells either during the process of deposition of nanoparticles or shortly after. Perhaps, the application of layer-by-layer (LBL) encapsulation technique, which is widely used for coating of various substrates, both planar surfaces and particles14-16 can be applied in order to produce a protective coating prior the deposition of nanoparticles. The LBL approach utilizes the consecutive placement of oppositely charged thin films of polyelectrolytes onto the surfaces, allowing almost unlimited number of bilayers to be formed.17 Various kinds of coating can be easily placed in between these multilayers, including proteins, nucleic acids, nanoparticles, etc.18-22 Cells are the suitable templates for coating with multilayered films. First, the cells were used only as sacrificial cores for the development of hollow microcapsules, as in the case of fixed human erythrocytes and bacterial cells.23,24 Next, the LBL approach was applied to encapsulate living yeast cells by the alternate adsorption of oppositely charged polyelectrolytes. It was found that after encapsulation the metabolic activity of coated cells was well preserved.25 Later, Krol et al. reported the creation of patterned surfaces on which both cell-friendly and passivated areas were present based on polyelectrolyte coating of both cells and surfaces; it is likely that the coatings could act as a protective shell for the cells in aggressive environments.26 Although many attempts have already been done, the modification of living cells with nanomaterials in order to develop novel immobilization techniques and microdevices is still a challenge. To our knowledge, there were no any reports so far describing the fabrication of thin polyelectrolyte films incorporating metal nanoparticles on microscopic fungal cell walls. The incorporation of nanoparticles as an element of (12) Li, Z.; Chung, S.; Nam, J.; Ginger, D. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2003, 42, 2306. (13) Sugunan, A.; Melin, P.; Schnurer, J.; Hilborn, J. G.; Dutta, J. Adv. Mater. 2007, 19, 77. (14) Dahne, L.; Peyratout, C. S. Angew. Chem., Int. Ed. 2004, 43, 3762. (15) Johnson, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F. Curr. Opin. Colloid Interface Sci. 2006, 11, 203. (16) Holt, B.; Lam, R.; Meldrum, F. C.; Stoyanov, S. D.; Paunov, V. N. Soft Matter 2007, 3, 188. (17) Decher, G. Science 1997, 277, 1232. (18) Hyde, K.; Rusa, M.; Hinestroza, J. Nanotechnology 2005, 16, S422. (19) Caruso, F.; Spasova, M.; Susha, A.; Giersig, M.; Caruso, R. A. Chem. Mater. 2001, 13, 109. :: (20) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mohwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (21) Park, M. K.; Deng, S.; Advincula, R. C. Langmuir 2005, 21, 5272. (22) Wang, Y.; Yu, A.; Caruso, F. Angew. Chem., Int. Ed. 2005, 44, 2888. :: :: (23) Baumler, H.; Neu, B.; Voigt, A.; Mitlohner, R.; Leporatti, S.; Gao, C. :: Y.; Donath, E.; Kiesewetter, H.; Mohwald, H.; Meiselman, H. J. J. Microencapsulation 2001, 18, 385. (24) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baumler, H.; Kiesewetter, H.; Mohwald, H. Chem.;Eur. J. 2002, 8, 5481. (25) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047. (26) Krol, S.; Nolte, M.; Diaspro, A.; Mazza, D.; Magrassi, R.; Gliozzi, A.; Fery, A. Langmuir 2005, 21, 705.
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polyelectrolyte multilayers allows the controllable modification of cells as templates and elements of microdevices. Using nanoparticles as components of LBL deposited polyelectrolyte coatings gives an advantage of modifying optical, electrical, magnetic, and some other properties of coated materials. Nanoparticles may be regarded as both functional (i.e., modified electric, optical, or magnetic properties) and structural (i.e., improved adhesion) elements. LBL approach provides us with a universal instrument to create an almost unlimited number of layers17 with varying polyelectrolytes and nanoparticles. In the case of living fungal cells it is crucial to provide the viability of the modified cells. In the current study we applied the LBL assembly of oppositely charged polyelectrolytes and gold and silver nanoparticles layerwise incorporated between polyelectrolyte layers aiming to obtain living fungi cells encapsulated into three-component shells. We used the cells of extensively used in several biotechnological applications unicellular Bakers’ yeast Saccharomyces cerevisiae and conidia of multicellular Trichoderma asperellum as the model organisms in order to investigate the process and effect of the placement of heterogeneous coatings onto these cells. Bakers yeast cells are widely used in various industrial and agricultural applications,27 whereas several Trichoderma strains serve as control agents against plant pathogens;28 therefore, the modification of these species might be of practical interest. Silver and gold nanoparticles along with synthetic and natural polyelectrolytes were successively deposited onto the cells, and the resulting shells were characterized using various microscopy techniques. Finally, the viability of the encapsulated cells was investigated, and they were further characterized using SERS.
Materials and Methods Chemicals and Cells. Poly(allylamine hydrochloride) (PAH,
Mw ∼ 15 000), sodium poly(styrene sulfonate) (PSS, Mw ∼ 70 000), and bovine serum albumin (BSA, Mw ∼ 56 000) were purchased from Sigma-Aldrich, UK; chlorauratic acid, silver nitrate, and sodium citrate were from Serva, Germany; DNA from chicken erythrocytes (∼50 kbp, as determined by 1% agarose gel electrophoresis) was from Reanal, Hungary. Other reagents were of analytical grade and used without purification. All experiments were carried out at room temperature; all solutions were prepared using double distilled water. S. cerevisiae yeasts were purchased from a local store. Before using, cells were suspended in water and washed three times in water followed by centrifugation (2 min, 2000 rpm). T. asperellum fungus was isolated from soil specimens obtained in Tatarstan and cultivated on the standard potato-agar media (homogenized potatoes, 200 g L-1; glucose, 20 g L-1; agar, 18 g L-1) for 5 days at +28 °C. After cultivation conidia were collected, washed three times in water in order to separate them from the remaining mycelium and stored at -4 °C. Synthesis of Nanoparticles. Gold nanoparticles (nanoAu) were obtained using the citrate reduction method,29 with minor alterations. Briefly, 220 mL of 0.02% (w/w) aqueous HAuCl4 was heated to boiling in a round-bottomed flask, and then 300 μL of 1% (w/w) aqueous sodium citrate was added rapidly while stirring and was further boiled for the next 0.5 h and then cooled and dialyzed in water for 3 days. Silver nanoparticles (nanoAg) (27) Demain, A. L.; Phaff, H. J.; Kurtzman, C. P. In The Yeasts. A Taxonomic Study, 4th ed.; Kurtzman, C. P., Fell, J. W., Eds.; Elsevier: Amsterdam, 1998; Vol. 13. (28) Ramot, O.; Viterbo, A.; Friesem, D.; Oppenheim, A.; Chet, I. Curr. Genet. 2004, 45, 205. (29) Cabib, E.; Roh, D.; Schmidt, M.; Crotti, L. B.; Varma, A. J. Biol. Chem. 2001, 276, 19679.
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were synthesized according to ref 30. Briefly, 250 mL of 0.01 M AgNO3 was heated at +90 °C, and then 40 mL of 1% (w/w) sodium citrate was added rapidly while stirring and incubated for 0.5 h; after cooling the nanoparticles were dialyzed in water for 3 days. Encapsulation of Cells. S. cerevisiae cells and T. asperellum conidia were coated with polyelectrolytes in a layer-by-layer fashion17 using alternating deposition of either PAH/PSS or BSA/DNA onto the surface of cells. PAH, PSS, and BSA were dissolved in 0.5 M NaCl at the concentration of 1 mg mL-1, and DNA was dissolved in 0.01 M Tris-EDTA buffer solution at the concentration of 100 μg mL-1. Taking into account the original negative surface charge of the cells, the procedure was started by coating with a positively charged polymer. 300 μL of the cells suspension was dropwise introduced into 1 mL of PAH or BSA solution during constant vortexing, incubated on a shaker for 15 min, and then centrifuged (2 min, 2000 rpm); the remaining excess of the polyelectrolyte was discarded, and the cells were redispersed and washed three times in water. Then the cells were similarly introduced into the solution of the respective polyelectrolyte (either PSS or DNA); the procedure was repeated to obtain PAH/PSS/PAH or BSA/DNA/BSA coatings on the cells. In the next step, the coated cells were introduced into a suspension of either gold or silver nanoparticles, incubated for 0.5 h, and washed with water; then two additional polyelectrolyte layers were deposited onto the cells. The resulting architecture of polyelectrolyte coating was as follows: PAH/ PSS/PAH/nanoparticles/PAH/PSS or BSA/DNA/BSA/nanoparticles/BSA/DNA. Characterization of Encapsulated Cells. Fungi cells coated with thin films incorporating metal nanoparticles were characterized by optical, scanning electron, scanning probe, and Raman microscopy. Bright field optical images of uncoated and coated cells were obtained using a Leica DM 1000 microscope equipped with 50 (numerical aperture 0.6) and 100 objectives (numerical aperture 1.25) and a Leica DFC 290 digital camera. Samples were imaged immediately after preparation. Scanning electron microscopy (SEM) images were obtained using a Carl Zeiss Evo-40 SEM. Samples of cells were prepared by dispersing 5 μL of a cellular suspension on the surface of specimen stubs and dried at +37 °C overnight. Then the cells were sputter-coated with a 10 nm thick gold layer using a Baltec SCD 005 sputter-coater. Scanning probe microscopy characterization of cells was performed using an NTERGA Prima atomic force microscope (AFM) (NT-MDT) equipped with NSG-3 cantilevers (force constant: 0.4-2.7 N m-1). Images were obtained at room temperature in air in tapping mode (resonance frequency: 81 kHz). In order to determine the root-mean-square roughness values, AFM images were processed with “Nova RC 1.0.26.578” software. Samples of cells were prepared by dispersing 5 μL of a cellular suspension on the surface of dust- and lipid-free glass coverslips and dried at +37 °C overnight. For transmission electron microscopy (TEM) bare and encapsulated cells were fixed in 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 samples were embedded in Epon resin (PELCO Eponate 12 Kit, Prod. No. 18010). Ultrathin sections were cut with diamond knife using a 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. TEM images were obtained using a Jeol 1200 EX microscope (Japan) operating at 80 kV. A Renishaw in Viva Reflex Raman microscopy system was used to perform all surface-enhanced Raman scattering (SERS) experiments. The system is automatically calibrated against silicon wafer peak at 520 cm-1. A diode laser at 830 nm and a
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50 (numerical aperture 0.75) objective with a laser power of 3 and 30 mW were used for polyelectrolytes mixed with silver nanoparticles and encapsulated cells measurements, respectively. The exposure time and the number of accumulation were 10 s and 1, respectively.
Results and Discussion In our study we used several widely used commercially available synthetic and natural oppositely charged polyelectrolyte pairs, namely PAH/PSS and BSA/DNA, in order to produce layered shells covering living fungi cells. We presumed that microbial cell might be coated with such films that will not seriously affect the viability of the cells; therefore, they can be applied as means for modification of living cells. The gold and silver nanoparticles used in this study were characterized prior to deposition using UV-vis spectroscopy and AFM (see Figure 1 in the Supporting Information). It was found that the average size distribution of the nanoparticles was 20 ( 4 nm for gold and 45 ( 7 nm for silver nanoparticles. The schematic representation of the formation of polyelectrolyte-nanoparticles shells is given in Figure 1. The procedure involves the deposition of positively charged polyelectrolytes PAH or BSA onto originally slightly negatively charged surfaces of cells, which provides the charge reversal, after which the second polyelectrolyte, PSS or DNA, was deposited onto the cellular surfaces likewise. Finally, another layer of positively charged polyelectrolyte, PAH or BSA, respectively to prior coatings, was placed, and the cells were ready to be coated with metal nanoparticles. Both gold and silver nanoparticles were synthesized using a citrate reduction method which produces highly monodisperse suspensions of nanoparticles.29 The negative surface charge of thus-synthesized nanoparticles is due to citrate ions adsorbed onto the particles and forming a stabilizing layer; therefore, these particles can be readily attached to the positively charged cells coated with PAH/PSS/PAH or BSA/DNA/ BSA. The adsorption of nanoparticles was followed by the deposition of an additional bilayer of either PAH/PSS or BSA/DNA; the final polyelectrolyte layer was negative to provide higher stability and prevent the adsorption of modified cells onto the walls of plastic tubes. BSA/DNA shells were used along with conventional PAH/PSS in order to investigate the possibility of application of this relatively cheap and biocompatible polyelectrolyte pair. The technique takes ∼3 h and yields in high quantities of encapsulated cells. The formation of nanoparticles incorporating polyelectrolyte shells on the surfaces of the cells can be clearly monitored even with the naked eye due to the characteristic changes in color of the suspensions of encapsulated cells in comparison with bare cells (see Figure 2 in the Supporting Information). Originally slightly yellow yeast cells obtained dark brown color after encapsulating with PAH/PSS shells containing silver nanoparticles and dark blue color if encapsulated with shells including gold nanoparticles. The same results were obtained for BSA/DNA shells (data not shown). It should be mentioned that the color of the suspensions is based only on those nanoparticles, which are incorporated into the shells because the low amounts of nanoparticles that were not adsorbed and remained free in the supernatant are removed during washing steps. The suspensions of conidia of T. asperellum are originally dark green; hence, it is not possible to see any visible changes in color after coating with shells, albeit the suspensions of coated conidia were found to be a bit darker than those of uncoated conidia. The optical Langmuir 2009, 25(8), 4628–4634
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Figure 1. Schematic representation of the encapsulation of living cells into the polyelectrolyte shells containing metal nanoparticles: (1) coating of intact cells with a triple layer of either PAH/PSS/PAH or BSA/DNA/BSA; (2) coating of PAH/PSS or BSA/DNA coated cells with either gold or silver nanoparticles; (3) coating of the cells with the final double layer of PAH/PSS or BSA/DNA. Structures are drawn not to scale. microscopy images (Figure 2) of cells coated with nanoparticles containing polyelectrolyte shells given together with the images of uncoated cells clearly show the formation of the shells which can be found out due to the changes of color in coated cells and the formation of aggregates of nanoparticles on the cellular surfaces. Both coated yeast cells and conidia became distinctly darker after encapsulation; the changes in color are due to incorporation of nanoparticles between polyelectrolyte layers. The effective light scattering by surface plasmons of gold and silver nanoparticles makes it possible to observe color change under a light microscope, making it possible to use optical microscopy as an inspection tool during the encapsulation process. It should be mentioned that the encapsulation of cells into shells without nanoparticles produces no visible changes in color in comparison with bare cells. Large aggregates of nanoparticles (approximately up to 1 μm in diameter) are likely to produce the aggregation of conidia into multicellular clusters, as can be seen in Figure 2g,h, although many single cells still can be found. Several reasons might cause such aggregation; we estimate that negatively charged nanoparticle aggregates can form bridges between oppositely charged cells, resulting in the formation of multicellular assemblies. The second reason might be the coating of initially existing aggregates of conidia with polyelectrolytes/nanoparticles. It was found that T. asperellum conidia are readily assembled into either spherical or amorphous multicellular structures (as presented in Figure 3 in the Supporting Information). This also explains the fact that encapsulated yeast cells are wellseparated with almost no aggregates formed in the cellular suspensions after coating with nanofilms and nanoparticles. Scanning electron microscopy was applied for the detailed imaging of the encapsulated cells. The typical images obtained are given in Figure 3. One can compare the cellular surface of nonmodified cells and conidia (Figure 3a,f) with encapsulated cells. Unambiguously, encapsulation of cells into the layers of either PAH/PSS or BSA/DNA polymer pairs provides the incorporation of nanoparticles into the shells. Rough and peaky features of the encapsulated cells are distinctly different from relatively smooth surfaces of the uncoated cells. Although the density of the coverage with nanoparticles is not visibly high, a number of single and aggregated nanoparticles can be seen on cellular surfaces. As is shown in Langmuir 2009, 25(8), 4628–4634
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Figure 3b,c, the daughter cells are not coated with nanoparticles; therefore, it might be concluded that budding happened after the encapsulation, indicating the cells viability after the treatment with nanoparticles. Atomic force microscopy was applied to visualize the surface structure of uncoated and encapsulated cells. The results for S. cerevisiae and T. asperellum are presented in Figure 4. It is clearly seen that initially smooth surfaces of the cells are strongly modified after encapsulation; numerous peaks and creases are formed in the case of using both types of nanoparticles and pairs of polyelectrolytes. As one can see in the AFM images depicting the cells encapsulated in shells doped with gold nanoparticles, they form dense layers on the surfaces of the cells, in comparison with silver nanoparticle-doped shells. Nanoparticle layers are likely to be formed regardless of the polyelectrolyte pairs used, indicating that BSA/DNA multilayers can be effectively used to produce nanoparticle coatings on the cellular surfaces. Experiments were performed in ambient conditions in air; therefore, it might be questionable whether the increased roughness of the cells is due to dehydration of cells during the drying. It is true for mammal cells, which have only cellular membrane partitioning the cytoplasm from the environment. However, we suppose that fungal cell wall, which acts as a protective barrier between the cell and the environment, provides the distinct cellular shape and size, prevents the dehydration of cells in hypotonic media,29 and even after drying preserves its shape and original topography. Taking into account the fact that samples of both bare and encapsulated cells in this study were prepared under the same conditions and there is no indication of any deformity in bare cells (additional AFM images of bare cells are given in the Supporting Information), we conclude that the increased surface roughness in encapsulated cells is due to nanoparticles incorporated into polyelectrolyte films. The comparison of average roughness parameters of bare and encapsulated cells is presented in the Supporting Information. Nanoparticles are the objects that can effectively increase the surface area and adhesion properties of the modified cells. On the contrary, they may affect viability of microorganisms. To preserve the viability of the cells encapsulated into the shells was the key feature of the current research. It was found that the yeast cells and T. asperellum conidia remained vital after the encapsulation in shells comprising both polyelectrolyte pairs (PAH/PSS and BSA/DNA) and nanoparticles (nanoAu and nanoAg) and were still able for budding (in case of yeast cells) and formation of hyphae (in case of T. asperellum). The encapsulated conidia were viable, as is shown in Figure 5a for the hyphae of Trichoderma asperellum conidia coated with PAH/PSS/PAH/nanoAg/PAH/PSS after the incubation. In contrast, noncoated conidia introduced into the as-made suspension of silver nanoparticles did not form mycelium even after a week of incubation at +37 °C in the aqueous sucrose (Figure 5b), pointing out that both gold and silver nanoparticles are a toxic environment for fungi cells. Numerous budding yeast cells were detected microscopically after adding 0.01 M aqueous sucrose into the suspensions of the encapsulated cells and subsequent incubation at +37 °C for 12 h (Figure 5c); the appearance of alcohol in the yeast suspension also indicated the intact enzymatic activity of coated yeast cells. On the contrary, no budding bare yeast cells were found after their incubation in the suspension of asmade silver nanoparticles and the subsequent incubation in sucrose solution for 1 week; it was noticed that cells were highly associated with aggregated nanoparticles (Figure 5b). DOI: 10.1021/la803871z
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Figure 2. Typical optical microscopy images of (a) bare yeast cells, (b) yeast cells coated with PAH/PSS/PAH/nanoAu/PAH/PSS, (c) yeast cells coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (d) yeast cells coated with BSA/DNA/BSA/nanoAu/BSA/DNA, (e) yeast cells coated with BSA/ DNA/BSA/nanoAg/BSA/DNA, (f) bare T. asperellum conidia, (g) T. asperellum conidia coated with PAH/PSS/PAH/nanoAu/PAH/PSS, (h) T. asperellum conidia coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (i) T. asperellum conidia coated with BSA/DNA/BSA/nanoAu/BSA/DNA, and (j) T. asperellum conidia coated with BSA/DNA/BSA/nanoAg/BSA/DNA.
Figure 3. Typical scanning electron microscopy images of (a) bare yeast cells, (b) yeast cells coated with PAH/PSS/PAH/nanoAu/PAH/PSS, (c) yeast cells coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (d) yeast cells coated with BSA/DNA/BSA/nanoAu/BSA/DNA, (e) yeast cells coated with BSA/DNA/BSA/nanoAg/BSA/DNA, (f) bare T. asperellum conidia, (g) T. asperellum conidia coated with PAH/PSS/PAH/nanoAu/PAH/ PSS, (h) T. asperellum conidia coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (i) T. asperellum conidia coated with BSA/DNA/BSA/nanoAu/ BSA/DNA, and (j) T. asperellum conidia coated with BSA/DNA/BSA/nanoAg/BSA/DNA. As was mentioned earlier, SEM data (Figure 3b) also revealed that the encapsulated yeast cells were dividing by budding, indicating their viability. Although we did not calculate the percentage of the matured conidia or budding yeast cells, it is clear that at least a considerable part of the cells is vital. Moreover, it was observed that after the longer periods of incubation almost all conidia formed mycelium. The same results were obtained for BSA/DNA-coated cells with shells doped with both gold and silver nanoparticles. This data indicates not only the biocompatibility and low (or no) cytotoxicity of polyelectrolyte shells doped with metal nanoparticles but also the elastic and permeable behavior of the walls of the shells, allowing budding and formation of hyphae, which needs the shells to be pierced in order to allow the separation of daughter cells and apical growth of hyphae. This probably reduces the rate of maturing, and further experiments are needed to investigate this. Taking into account the viability of encapsulated cells, we assume that the polyelectrolyte films between the nanoparticles act as a barrier between toxic metal nanocrystals and cellular walls, preventing their intracellular uptake and the toxic effects onto cells. In order to further investigate the distribution of nanoparticles on the surfaces of polyelectrolyte-modified cells as well 4632
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as to find out whether the nanoparticles penetrate the cell walls, TEM images of bare and encapsulated cells were obtained. As one can see in Figure 6, transmission electron microscopy clearly demonstrates the immobilization of both single and aggregated nanoparticles on the cell walls of encapsulated cells. The density of nanoparticles coating revealed with TEM corresponds well with SEM and AFM images. Thin sections of encapsulated cells show no indication of penetration of nanoparticles inside the cells, which might explain the viability of encapsulated cells in presence of cytotoxic nanoparticles. Indeed, electrostatically preserved assembly of polyelectrolytes may effectively prevent the penetration of nanoparticles, at least of sizes which were used in this study. Moreover, polyelectrolytes preserve the integrity of nanoparticles layers even under mechanical stress (shaking, etc.). The modification of living microbial cells with nanofilms and nanoparticles might be utilized in a number of applications, among which we are pointing out such as the controllable adhesion of cells onto various surfaces (e.g., the improved adhesion of bacteria onto the surface of intestines in order for the treatment of disbacteriosis, etc.), the spatial orientation of modified cells in the electric or magnetic field, etc. These applications are currently under investigation by Langmuir 2009, 25(8), 4628–4634
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Figure 4. Atomic force microscopy images of (a) bare yeast cells, (b) yeast cells coated with PAH/PSS/PAH/nanoAu/PAH/PSS, (c) yeast cells coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (d) yeast cells coated with BSA/DNA/BSA/nanoAu/BSA/DNA, (e) yeast cells coated with BSA/ DNA/BSA/nanoAg/BSA/DNA, (f) bare T. asperellum conidia, (g) T. asperellum conidia coated with PAH/PSS/PAH/nanoAu/PAH/PSS, (h) T. asperellum conidia coated with PAH/PSS/PAH/nanoAg/PAH/PSS, (i) T. asperellum conidia coated with BSA/DNA/BSA/nanoAu/BSA/DNA, and (j) T. asperellum conidia coated with BSA/DNA/BSA/nanoAg/BSA/DNA. Left to right: 3D reconstructions of a single cell; 1 1 μm highresolution images of the cellular surface of the respective cell. our group. The selective detection of microorganisms is one of the practically important tasks, which also can be realized via the proposed method of the microbial modification. In this study we used SERS to demonstrate the applicability of the described technique for enhancing Raman scattering of encapsulated cells. SERS can provide chemical information about close surroundings of gold or silver nanoparticles. The SERS studies of bacterial cells using colloidal silver and gold nanoparticles revealed that the information gained through the SERS spectra is mostly from bacterial cell (30) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757. (31) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391. (32) Efrima, S.; Bronk, B. V. J. Phys. Chem. B 1998, 102, 5947. (33) Zeiri, L.; Bronk, B.; Shabtai, V. Y.; Eichler, J.; Efrima, S. Appl. Spectrosc. 2004, 58, 33.
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wall.30-38 The charges carried on the bacterial cell wall due to the functional groups such as COO- and -NH+ 2 define the mode of interaction of bacteria cell wall and noble metal nanoparticles.37,38 In addition, the presence of -SH-containing residues further enhances this interaction. Therefore, the SERS spectra obtained from the noble metal nanoparticle-coated cells can provide valuable information. The T. (34) Zeiri, L.; Efrima, S. J. Raman Spectrosc. 2005, 36, 667. (35) Sengupta, A.; Laucks, M. L.; Davis, E. J. Appl. Spectrosc. 2005, 59, 1016. (36) Sengupta, A.; Mujacic, M.; Davis, E. Anal. Bioanal. Chem. 2006, 386, 1379. (37) Kahraman, M.; Yazici, M. M.; Sahin, F.; Culha, M. J. Biomed. Opt. 2007, 12, 054015. (38) Kahraman, M.; Yazici, M. M.; Sahin, F.; Bayrak, O. F.; Culha, M. Appl. Spectrosc. 2007, 61, 479.
DOI: 10.1021/la803871z
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Figure 5. Optical microscopy images of (a) matured T. asperellum conidia (encapsulated in shells comprising PAH/PSS/PAH/nanoAg/ PAH/PSS) and hyphae after the incubation at +37 °C for 12 h, (b) nonmatured bare T. asperellum conidia after the incubation for 12 h in the suspension of as-made silver nanoparticles and the subsequent incubation at +37 °C in the 0.01 M aqueous sucrose solution for 1 week, (c) budding yeast cells (encapsulated in shells comprising PAH/ PSS/PAH/nanoAg/PAH/PSS) after the incubation at +37 °C for 12 h in the 0.01 M aqueous sucrose solution, and (d) nonbudding yeast cells after the incubation for 12 h in the suspension of as-made silver nanoparticles and the subsequent incubation at +37 °C in the 0.01 M aqueous sucrose solution for 1 week.
asperellum conidia or aggregates of conidia composed of a few spores were large enough to be located under an optical microscope of the Raman microscopy system to focus the laser light onto them. The SERS spectra obtained from T. asperellum conidia gold and silver nanoparticles embedded through four different assays are presented in Figure 6 in the Supporting Information along with related discussion. We suppose that the technique described in the current paper might find an application in the identification and characterization of microorganisms using SER.
Conclusions In this paper, living fungi cells were encapsulated layerwise with several consecutive polyelectrolyte thin films and with metal nanoparticles incorporated between these films. The encapsulated cells were investigated using various microscopic techniques and SERS. Gold and silver nanoparticles were successfully immobilized on the surface of the cells, effectively altering the color and the surface topography of the encapsulated cells. Both gold and silver nanoparticlecoated cells remained viable after the encapsulation, indicating the protective function of polyelectrolyte coating against aggressive nanoparticle influence. The increased surface roughness of the encapsulated cells can be utilized in processes
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Figure 6. Typical transmission electron microscopy images of (a) bare yeast cells, (b) yeast cells coated with PAH/PSS/PAH/nanoAu/ PAH/PSS, (c) yeast cells coated with PAH/PSS/PAH/nanoAg/PAH/ PSS, (d) bare T. asperellum conidia, (e) T. asperellum conidia, and (f) T. asperellum conidia coated with PAH/PSS/PAH/nanoAg/PAH/ PSS. of controlled immobilization of the cells on various surfaces, especially in cases when a high level of adsorption is desired. We suppose that the method described here to coat living fungi cells might be applied successfully for the modification of other microbial cells for their modification and characterization. Acknowledgment. The authors thank Prof. Dr. D. G. Ishmuchametova, Prof. Dr. M. K. Salakhov, Prof. Dr. F. Sahin, and Prof. Dr. A. P. Kiyasov for their assistance, Mrs. K. Tatlidil for SEM images, Dr. V.V. Salnikov for TEM images, and two anonymous reviewers for their valuable comments. The financial support of Yeditepe University is gratefully acknowledged. Supporting Information Available: AFM images of nanoparticles used, UV-vis spectra of nanoparticles, pictures of suspensions of intact and encapsulated cells, optical microscopy images of intact single conidia and conidia aggregates, AFM images of bare yeast cells and T. asperellum conidia, average roughness values for bare and encapsulated cells, and SESR spectra of encapsulated cells and polyelectrolytes. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2009, 25(8), 4628–4634
