Microfabrication of Patterns of Adherent Marine Bacterium

Apr 16, 2010 - ... School of Chemistry, Clayton Campus, Victoria 3800, Australia. ..... scan rates (without the loss of data points) and lower optical...
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Microfabrication of Patterns of Adherent Marine Bacterium Phaeobacter inhibens Using Soft Lithography and Scanning Probe Lithography Chuan Zhao,*,†,‡ Malte Burchardt,†,§ Thorsten Brinkhoff,‡ Christine Beardsley,‡ Meinhard Simon,‡ and Gunther Wittstock† †

Department of Pure and Applied Chemistry, Center of Interface Science and ‡Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University of Oldenburg, 26111 Oldenburg, Germany. §Present address: Malte Burchardt, Fraunhofer Institute for Manufacturing Technology and Applied Materials Research, Wiener Strasse 12, D-28325 Bremen, Germany Received December 16, 2009. Revised Manuscript Received February 26, 2010

Two lithographic approaches have been explored for the microfabrication of cellular patterns based on the attachment of marine bacterium Phaeobacter inhibens strain T5. Strain T5 produces a new antibiotic that makes this bacterium potentially interesting for the pharmaceutical market and as a probiotic organism in aquacultures and in controlling biofouling. The microcontact printing (μCP) method is based on the micropatterning of self-assembled monolayers (SAMs) terminated with adhesive end groups such as CH3 and COOH and nonadhesive groups (e.g., short oligomers of ethylene glycol (OEG)) to form micropatterned substrates for the adhesion of strain T5. The scanning probe lithographic method is based on the surface modification of OEG SAM by using a microelectrode, the probe of a scanning electrochemical microscope (SECM). Oxidizing agents (e.g., Br2) were electrogenerated in situ at the microelectrodes from Br- in aqueous solution to remove OEG SAMs locally, which allows the subsequent adsorption of bacteria. Various micropatterns of bacteria could be formed in situ on the substrate without a prefabricated template. The fabricated cellular patterns may be applied to a variety of marine biological studies that require the analysis of biofilm formation, cell-cell and cell-surface interactions, and cell-based biosensors and bioelectronics.

Introduction Research on bacterial adhesion is a large field covering different aspects such as biofilm formation, fundamental microbiology, marine science, soil and plant ecology, food safety, and biomedical problems.1-4 The mechanism of bacterial adhesion is indeed very complicated.4-8 One of the main reasons is that bacteria are always surrounded in vivo by a complex spatiotemporal matrix. They have to interact with and respond to underlying substrates, neighboring cells, the surrounding extracellular matrix, soluble factors, and other local physical forces.4-8 Driven by the concept of lab on a chip, new tools based upon microfabrication technology and surface chemistry are being developed that allow greater control over the spatial organization and temporal presentation of cellular microenvironments.9-11 Fundamentally, it offers new opportunities and advantages for mechanistic studies of bacterial adhesion by the separate variation *Corresponding author. Present address: Monash University, School of Chemistry, Clayton Campus, Victoria 3800, Australia. Tel: þ61 3 99051520. Fax: þ61 3 99054597. E-mail: [email protected].

(1) Dankert, J.; Hogt, A. H.; Feijen, J. Crit. Rev. Biocompat. 1986, 2, 219–301. (2) O’Toole, G.; Kaplan, H. B.; Kolter, R. Annu. Rev. Microbiol. 2000, 54, 49–79. (3) Allison, D. G.; Gilbert, P.; Lappin-Scott, H. M.; Wilson, M. Community Structure and Co-Operation in Biofilms; Cambridge University Press: Cambridge, U.K., 2000; p 349. (4) Watnick, P.; Kolter, R. J. Bacteriol. 2000, 182, 2675–2679. (5) Dougherty, S. H. Rev. Infect. Dis. 1988, 10, 1102–1117. (6) Dougherty, S. H.; Simmons, R. L. Infect. Dis. Clin. North Am. 1989, 3, 199–209. (7) Gristina, A. G. Science 1987, 237, 1588–1595. (8) Katsikogianni, M.; Missirlis, Y. F. Eur. Cells Mater. 2004, 8, 37–57. (9) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–411. (10) Li, N.; Tourovskaia, A.; Folch, A. Crit. Rev. Biomed. Eng. 2003, 31, 423–488. (11) Raghavan, S.; Chen, C. S. Adv. Mater. 2004, 16, 1303–1313.

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of each stimulus of the complex microenvironments, for example, the biochemical composition and topography of the substrate, the medium composition, neighboring cells, and so forth.9-14 Bacterial cells have been extensively studied for sensing applications because analyte specificity can be achieved easily by genetic engineering and because of the robust nature of these microorganisms compared to that of mammalian cells. The ability to attach bacteria selectively to micropatterned substrates could be used to create sensor arrays for an application such as fast, low-cost in vitro screening under a large number of conditions.9,10,15-17 From a practical point of view, the control of bacterial adhesion could also be a valuable tool toward developing antifouling materials. Bacterial adhesion is controlled by the chemical and physical characteristics of surfaces such as wettability, charge, roughness, and so forth.18-20 These factors can be readily modified to control the spatial distribution of cells on a substrate. The most commonly used method in micropatterning cells is to define and control regions that promote or resist the adhesion of cells by patterning cell-adhesive and cell-repellent regions on the surface (12) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267–273. (13) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992–5996. (14) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425–1428. (15) Cowan, S. E.; Liepmann, D.; Keasling, J. D. Biotechnol. Lett. 2001, 23, 1235–1241. (16) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336–6343. (17) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828–2834. (18) Jung, D. R.; Kapur, R.; Adams, T.; Giuliano, K. A.; Mrksich, M.; Craighead, H. G.; Taylor, D. L. Crit. Rev. Biotechnol. 2001, 21, 111–154. (19) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227–256. (20) Ito, Y. Biomaterials 1999, 20, 2333–2342.

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using soft lithography (e.g., microcontact printing (μCP) patterns of self-assembled monolayers).9-12,21 Normally, a transparent elastomer such as poly(dimethylsiloxane) (PDMS) is used as a stamp or mold with which a cytophilic pattern is formed on a substrate. The empty area is then filled with an inert SAM to prevent nonspecific adhesion. SAMs that are terminated in short oligomers of the ethylene glycol (OEG) unit (HS(CH2)11(OCH2CH2)nOH; n = 3-7) were most often used in these studies to resist the nonspecific adsorption of proteins or cells.22-24 OEG SAMs are proven to be very effective nonadsorption surfaces and have been used as the “standard” inert surfaces for protein and cell adsorption. Despite the immense practical importance of the protein and the cell resistance effect of OEG SAMs, the underlying mechanisms are not completely clear. Herrwerth et al. summarized the various factors that are critical for the cellrepellent properties of such monolayers and suggested that only the combination of three factors, namely, the internal hydrophilicity, the terminal hydrophobicity, and the lateral packing density, allows the formation of a SAM that is fully protein/ cell-resistant.24 If one of these factors is unfavorable or absent, then the overall protein/cell resistance decreases. Scanning probe lithography is another class of surface-patterning techniques using the probe in scanning probe microscopy (SPM) as a surface-modification tool.25 Scanning electrochemical microscopy (SECM), one kind of SPM, is widely used for electrochemical surface modification on the micrometer or submicrometer scale.26,27 SECM is based on scanning an ultramicroelectrode (UME) close to a surface immersed in an electrolyte solution containing electroactive species. So far, SECM has been successfully used to drive the etching of metals and semiconductors as well as in the deposition of metals, metal oxides, and to some extent biological substances, polymers, and cells.28-33 Kaji et al. reported the use of SECM to produce Br2/OBr- to denature a bovine serum albumin-coated substrate, which allows subsequent cell attachment and patterning.32,33 Recently, we observed that the “standard” nonadsorption surface, OEG SAM, could be made adsorptive by exposure to electrogenerated Br2 from aqueous solution.34 The electrogenerated bromine removes the oligo(ethelyene glycol) part of the monolayer on a second timescale and exposes the CH3-terminated alkyl chain of the SAM to the surface that is known to be cell-adhesive. This observation led to a microelectrochemical method to micropatterning mamallian cells in situ on SAM surfaces. We also demonstrated that the method could be integrated into a widely used microcontact printing (21) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575. (22) Ballav, N.; Thomas, H.; Winkler, T.; Terfort, A.; Zharnikov, M. Angew. Chem., Int. Ed. 2009, 48, 5833–5836. (23) Montague, M.; Ducker, R. E.; Chong, K. S. L.; Manning, R. J.; Rutten, F. J. M.; Davies, M. C.; Leggett, G. J. Langmuir 2007, 23, 7328–7337. (24) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359–9366. (25) Kr€amer, S.; Fuierer, R. R.; Gorman, C. B. Chem. Rev. 2003, 103, 4367– 4418. (26) Mandler, D. Micro- and Nanopatterning Using the Scanning Electrochemical Microscope. In Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 593-627. (27) Wittstock, G.; Burchardt, M.; Pust, S. E.; Shen, Y.; Zhao, C. Angew. Chem., Int. Ed. 2007, 46, 1584–1617. (28) Radtke, V.; Heinze, J. Z. Phys. Chem. 2004, 218, 103–121. (29) Borgwarth, K.; Rohde, N.; Ricken, C.; Hallensleben, M. L.; Mandler, D.; Heinze, J. Adv. Mater. 1999, 11, 1221–1226. (30) Turyan, I.; Krasovec, U. O.; Orel, B.; Saraidorov, T.; Reisfeld, R.; Mandler, D. Adv. Mater. 2000, 12, 330–333. (31) Mandler, D.; Bard, A. J. J. Electrochem. Soc. 1990, 137, 1079–1086. (32) Kaji, H.; Tsukidate, K.; Matsue, T.; Nishizawa, M. J. Am. Chem. Soc. 2004, 126, 15026–15027. (33) Kaji, H.; Kanada, M.; Oyamatsu, D.; Matsue, T.; Nishizawa, M. Langmuir 2004, 20, 16–19. (34) Zhao, C.; Witte, I.; Wittstock, G. Angew. Chem., Int. Ed. 2006, 45, 5469– 5471.

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method combining the advantages of high throughput from microcontact printing and maskless in situ manipulation by a scanning probe.35 An investigation of substrates immersed as electrodes of a galvanic cell mimicking the situation in SECM surface modification followed by a structural analysis of the monolayer prepared from selectively deuterated OEG-terminated SAMs showed the fast degradation of the OEG part of the molecule.36 The remaining (defective) monolayer is removed very slowly because heterogeneous electron transfer from the gold substrates occurs via the defects and scavenges the Br2 reagents. This process restricts the lateral spreading of the modified regions and leads to a robust, well-controlled procedure.36 In this study, both μCP and SECM methods have been explored for micropatterning Phaeobacter inhibens (strain T5), a gram-negative marine bacterium isolated from the German Wadden Sea. This recently discovered strain can produce a new antibiotic named tropodithietic acid and is potentially very interesting for the pharmaceutical market and for controlling biofouling by other microorganisms on submerged T5-colonized surfaces.37 Furthermore, controlled settlement and enrichment of Phaeobacter spp. is important because these organisms are of interest for use in aquacultures as antagonists against fish and scallop pathogenic bacteria such as Vibrio anguillarum.38 Therefore, the microfabricated cellular patterns based on strain T5 could be useful for whole cell-based sensor devices. Importantly, these micropatterning techniques may bring about new opportunities and advantages for fundamental bacterial adhesion studies by providing greater control over the spatial organization and temporal presentation of bacterial microenvironments. The advantages and disadvantages of the two techniques are discussed in this study.

Materials and Methods Microcontact Printing of SAMs. Gold films as substrates for the formation of SAMs are prepared on microscope glass slides cleaned by immersion in piranha solution prepared by mixing 70% (v/v) concentrated H2SO4 with 30% H2O2 for 20 min to 1 h, followed by thoroughly rinsing the slides in deionized water and drying them in a stream of nitrogen. (Caution! Piranha solution is a powerful oxidizer and can react violently with organic material. It should be stored in containers that prevent pressure buildup.) The samples were then placed into the evaporation chamber of a metal evaporator (Tectra GmbH, Frankfurt, Germany). The system was evacuated to 2 μm could also be observed on the substrate surface. Inspection at higher magnification (Figure 1b) reveals the porous nanostructures of the cell wall, presumably composed of phospholipids and negatively charged lipopolysaccharides typical of a gram-negative cell.46 The μCP method has been routinely used to pattern mammalian cells. Nevertheless, it has rarely been used for marine bacteria. In this study, the μCP method was first explored for micropatterning strain T5 via two routes (Figure S1 in Supporting Information). Route A used a procedure similar to that often used for mammalian cells.10-12 A PDMS stamp with micrometer-scale features was first inked with CH3-thiol (cytophilic) and stamped onto a gold substrate. The hexadecanethiol molecules were then transferred to the substrate to form a hydrophobic pattern. The empty space on the gold substrate was refilled with OEG-thiol to form a surface that is inert to protein adsorption and bacterial adhesion. SAMs terminated with CH3 allow protein adsorption. An extracellular protein layer was then adsorbed onto the CH3terminated region of the SAM to promote the adhesion of bacteria in subsequent cell seeding. Route B is based on our previous test of cell counting after the exposure of T5 cultures to different SAM-coated surfaces.47 Thus, SAMs with favorable surface chemistry for T5 adhesion were directly used for cell seeding without an extracellular protein adsorption step. It has been found that strain T5 preferentially adhered to COOH-terminated SAM surfaces in less than 10 min. Therefore, as shown in Figure S1, a PDMS stamp was inked with COOH-terminated 12-mercaptodecanoic acid. Hydrophilic micropatterns of COOH-terminated SAMs were transferred using μCP, and the empty space was then filled with HS(CH2)11(OCH2-CH2)6OH in order to resist the nonspecific adsorption of proteins or bacteria. The micropatterned SAM substrates were characterized by using pulse force-mode atomic force microscopy with functionalized tips. PFM-AFM is chosen because it offers faster scan rates (without the loss of data points) and lower optical interference than lateral force microscopy, which has often been used in (46) Martens, T.; Heidorn, T.; Pukall, R.; Simon, M.; Tindall, B. J.; Brinkhoff, T. Int. J. Syst. Evol. Microbiol. 2006, 56, 1293–1304. (47) Zhao, C.; Brinkhoff, T.; Burchardt, M.; Simon, M.; Wittstock, G. Ocean Dyn. 2009, 59, 305–315.

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characterizing patterned SAMs.48 Because adhesive forces are directly related to the interaction between chemical functional groups at the tip and sample surfaces, mapping of adhesion forces by PFM-AFM was combined with chemically modified tips (COOH-SAM) to image a sample surface with chemical sensitivity (Figure 2a). In Figure 2b, the micropatterned OEG (bright region) and CH3 (dark region) SAMs fabricated by μCP could clearly be discriminated in the adhesion force image without any optical interference. Adhesive forces were higher (bright) for the hydrophilic OEG regions than for the hydrophobic CH3-SAM region (dark) using the hydrophilic tip. This is due to the hydrophilic-hydrophilic interaction, which is generally larger than the hydrophilic-hydrophobic and hydrophobichydrophobic interactions.25 It should be noted that the adhesive region visualized by PFM-AFM is attributed to the hydrophilichydrophilic interaction between the tip and the OEG region. This is a different meaning than that used for the cell-adhesive region when talking about bacterial adhesion, which also involves other interactions and forces. Actually, the OEG were totally inert for the following bacterial adhesion. Simultaneously acquired topographic and stiffness images of a patterned surface (not shown) did not show the pattern because the tiny height difference between both SAMs was too small to be resolved on a polycrystalline Au substrate. To promote the adhesion of bacteria, an adhesive layer of extracellular protein, fibronogen, has been introduced onto micropatterned CH3-terminated SAM surfaces. A number of studies showed that adsorbed fibrinogen promotes not only the specific adherence of mammalian cells but also that of bacteria.49-51 The adsorption of the protein was characterized by CLSM after incubating a patterned CH3/OEG substrate in 100 μg/mL fluorescently labeled fibrinogen-Alexa 488 in 0.1 M PBS buffer (pH 7.4) for 4 h. Figure 3 shows that the proteins exclusively absorbed on the CH3-terminated regions (bright) whereas no adsorption was observed for the OEG-terminated regions (dark). The incubation of the slides as prepared from routes A and B (Figure S1) in bacterial culture allows the adherence of strain T5 (48) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071–2073. (49) Flemming, R. G.; Lai, Q. J.; Herrmann, M.; Mosher, D. F.; Proctor, R. A.; Cooper, S. L. Trans. Soc. Biomater. 1993, 16, 153. (50) Herrmann, M.; Lai, Q. J.; Albrecht, R. M.; Mosher, D. F.; Proctor, R. A. J. Infect. Dis. 1993, 167, 312–322. (51) Paulsson, M.; Kober, M.; Freij-Larsson, C.; Stollenwerk, M.; Wesslen, B.; Ljungh, A. Biomaterials 1993, 14, 845–853.

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Figure 3. Scanning confocal fluorescence micrographs of the adsorption of extracellular protein onto micropatterned SAMs using μCP. Fluorescently labeled protein of fibrinogen-Alexa 488 selectively adsorbed into CH3-terminated SAM regions (bright part). The dye was excited at a wavelength of 488 nm, and the fluorescence was detected over a spectral range of 500-535 nm.

Figure 4. Phase-contrast micrographs of the formation of micropatterns of marine bacteria using μCP. (A) Attachment of strain T5 after 30 min of incubation in cell cultures (107 cells/mL). (B) Phase-contrast micrographs of the micropatterned cell after 12 h of incubation in a medium at 20 °C without shaking.

onto the surface and the formation of micropatterns after 30 min of incubation in a T5 culture. As shown in Figure 4, bacteria selectively adhered to the CH3/protein-coated or COOH-coated regions, and OEG effectively blocked bacterial adherence (Figure 4A). No obvious difference was observed for the CH3/ protein-coated and COOH-coated substrates regarding the cell density, indicating that both specific ligand-receptor interactions and hydrophobic interactions play roles in the adhesion process of strain T5. The attached bacteria underwent proliferation and occupied the adhesive region completely if further incubated in complete medium marine broth 2216 at room temperature for another 12 h (Figure 4B). In summary, the marine bacteria can be patterned onto substrates by using μCP with high throughput. Both routes work well. Normally, micropatterned SAMs with a hydrophilic end group (e.g., COOH) is favorable to bacterial adhesion. Therefore, such a surface could be directly exposed to a bacterial culture. Micropatterned SAMs with a hydrophobic end group (e.g., CH3) and a subsequently adsorbed extracellular matrix protein layer provided an adhesive surface and would be useful for studying specific bacteria-substrate interactions. Micropatterning of Strain T5 Using SECM. An alternative template-free in situ method was developed for micropatterning bacteria using the surface-modification function of SECM.34 This Langmuir 2010, 26(11), 8641–8647

method is based on our recent finding that the cytophobic nature of the OEG SAM can be rapidly switched to cell-adhesive by exposure to oxidizing agents such as Br2, which can be electrogenerated from Br- in aqueous solution at an ultramicroelectrode (UME) attached to an SECM.34 Previous studies have provided evidence that Br2 actually quickly removes the functional oligo(ethelyene glycol) part of the monolayer, leading to an increase in external hydrophobicity.35,36 Micropatterns of bacteria could be formed in situ by scanning a UME closely above the OEG SAM substrate. The dimensions of the UME govern the lateral resolution of the pattern. In principle, such electrodes can be of nanometer dimensions whereas no restriction exists for larger sizes. Electrochemical modification was performed by using a microelectrode with a radius of rT = 12.5 μm placed 5 μm above the substrate (Figure 5a). A 5 s potential pulse of ET = 1.2 V was applied to the microelectrode in 0.1 M phosphate buffer containing 25 mM KBr (pH 7.4). The electrochemically generated Br2 diffused to the substrate and quickly reacted with the OEG monolayer locally (Figure 5b). Although spots could be formed as a result of holding the microelectrode in a fixed location above the surface, lines could be generated by translating the microelectrode horizontally while producing Br2. The microelectrode potential was kept at 1.2 V during “drawing” and at 0 V while not DOI: 10.1021/la904725g

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drawing. When the microelectrochemically patterned substrates were incubated in extracellular protein solution, the protein adsorbed into the modified regions of the OEG substrate and the unmodified region of OEG retained its protein-repelling

Figure 5. SECM method for template-free micropatterning of marine bacteria. (a) The OEG SAM was locally modified by using microelectrochemically generated Br2. (b) Extracellular protein selectively adsorbed into modified regions. (c) Protein promotes cell adhesion in the subsequent bacterial seeding.

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properties. The micropatterned proteins will promote the adhesion of strain T5, resulting in micropatterns of bacteria. Various methods have been used to characterize the microelectrochemically formed patterns. This step is crucial because the whole method will otherwise run in a “black box”. Without labeling, it is very difficult to discriminate the patterns using optical microscopy or AFM because of the tiny differences in height, surface hydrophobicity, and optical properties before and after modification on polycrystalline substrates. SECM has proven to be a powerful tool not only for surface modification but also for in situ characterization. The modification quality could be checked in situ by using SECM as an imaging tool. After the modification, an SECM image was recorded immediately in feedback mode using [Ru(NH3)6]3þ as the mediator (Figure 6a). The image contrast is based on the permeability difference of the SAM before and after treatment. At the unmodified SAM, the diffusion of [Ru(NH3)6]2þ to the underlying gold substrate is effectively blocked (Figure 6a). If the UME moved to the modified region of the monolayer, then the increased density of defects would cause the permeability of the SAM that allows [Ru(NH3)6]2þ to reach the gold surface where [Ru(NH3)6]2þ is oxidized back to [Ru(NH3)6]3þ. Regenerated [Ru(NH3)6]3þ diffuses back to the UME and causes an increase in the reduction current at the microelectrode (Figure 6b). Figure 6c shows SECM feedback images recorded after the micropatterning by local Br2 generation. The dark parts in the image indicate a higher reduction current and higher permeabilities. The adsorption of the protein at the electrochemically modified substrates was characterized by CLSM after incubating the substrate in 100 μg/mL fluorescently labeled fibrinogen (fibrinogenAlexa 488) in 0.1 M PBS buffer (pH 7.4) for 4 h (Figure 7). The exposure of the CH3-terminated alkyl chain of the OEG SAM allows the proteins to absorb exclusively onto the Br2-treated regions (bright) whereas no adsorption was observed for the OEG regions (dark). Furthermore, in comparison to the SECM images (spots in Figure 6), the fluorescence micrograph (Figure 7)

Figure 6. Characterization of the microfabricated OEG substrate for the micropatterning of bacteria using SECM. (a) Schematic for SECM feedback imaging of the modified OEG monolayer. Negative feedback was obtained with the OEG monolayer. (b) Positive feedback was obtained with the modified region of the monolayer because of the increased permeability. (c) SECM feedback images of microfabricated patterns on the OEG SAM substrate. The images were recorded using [Ru(NH3)6]3þ as the electron-transfer mediator in 0.1 M PBS buffer (pH 7.4) after SECM microfabrication. The higher current (dark regions) in the images indicates a higher permeability of the monolayer. ET = -400 mV, rT = 25 μm, d = 5 μm, and translation speed = 10 μm/s. 8646 DOI: 10.1021/la904725g

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Figure 7. Scanning confocal fluorescence micrographs of the adsorption of extracellular protein onto micropatterned SAMs using SECM. The fluorescently labeled protein of fibrinogen-Alexa 488 was selectively adsorbed into CH3-terminated SAM regions (bright part). The dye was excited at a wavelength of 488 nm, and the fluorescence was detected over a spectral range of 500-535 nm.

Figure 8. Scanning confocal reflection micrographs of the formation of micropatterns of marine bacteria using SECM. Attachment of strain T5 after 120 min of incubation in cell cultures (107 cells/mL).

indicates that the protein preferentially absorbed onto the middle of the modified area within the incubation time applied in this study. This is expected because the protein-resistant property of the OEG SAM is most effectively removed in the middle of the spot as a result of the Br2 concentration gradient. This could also be seen in the SECM images where the highest feedback current (highest destruction to the OEG layer) was observed in the middle part of the spot. The absorbed protein promotes bacterial adhesion during subsequent incubation in a T5 culture, allowing the formation of the bacterial micropatterns (Figure 8). Compared to the patterns seen in the SECM images (Figure 6) and the CLSM images (Figure 7), it can be confirmed that spots and linear patterns of adhered bacteria could be formed very flexibly by scanning the UME probe over the surface. Figure 8 shows that some spots have a relatively higher cell density than others, presumably because of the variation in microelectrochemical modification and subsequent protein adsorption processes. The bacterial pattern has a rather random internal structure and distribution (single spot in Figure 8). However, a clear edge between the modified and unmodified OEG regions was usually observed. A careful examination of the cell pattern (single spot in Figure 8) also reveals that the shape and the size of the cellular spot closely fit the geometry of the UME. This indicates that the lateral resolution of the pattern was mainly controlled by the dimension of the active area of the probe. Thus, bacterial patterns of higher resolution could in principle be obtained by using a smaller UME. Indeed, this has been demonstrated in other applications where the smallest surface patterns formed by UMEs are ca. 50 nm.28 Furthermore, it has been observed that UME-generated Br2 can be scavenged by heterogeneous electron transfer from gold during the modification once the OEG SAM becomes permeable. Thus, the extension of the modified regions and the subsequent bacterial patterns are also self-limited.36

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Conclusions A range of cellular micropatterns based on new marine strain T5 have been fabricated using μCP and SECM methods. Although μCP is suitable for producing extended, complex patterns of adhering bacteria cells for high-throughput applications, the microelectrode-based method is suitable for producing and subsequently manipulating micropatterns in situ, which will be important in fundamental studies. Ideally, a combination of both methods, namely, the formation of a large periodic pattern first by using μCP and subsequent modification of the existing pattern by using an ultramicroelectrode, would provide a versatile tool for forming more complex patterns for fundamental studies of intercellular interactions and heterotypic signaling in biofilm formation on surfaces and many other studies in microbiology based on the in situ control of the bacteria-substrate interaction. A variety of OEG- or PEG-functionalized materials such as polymers have been recently developed as coatings to reduce bacterial adhesion in a hygienic context. Our results also show that the fast reaction of OEG surfaces with oxidizers must also be considered when such surfaces additionally come into contact with oxidizing disinfectants. In such a situation, the disinfectant may quickly remove the cell-repellent property of the surface. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (Wi 1617/6) within the Research Group “Biogeochemistry of Tidal Flats”. Supporting Information Available: Schematic drawing showing the two routes for micropatterning marine bacterium strain T5 using μCP. This material is available free of charge via the Internet at http://pubs.acs.org.

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