Bacterial surface-layer proteins for electrochemical nanofabrication

Electrochimica Acta 53 (2007) 193–199

Bacterial surface-layer proteins for electrochemical nanofabrication Daniel B. Allred a , Mehmet Sarikaya a,b , Franc¸ois Baneyx a , Daniel T. Schwartz a,b,∗,1 a

b

Chemical Engineering Department, University of Washington, Seattle, WA 98195-1750, USA Materials Science & Engineering Department, University of Washington, Seattle, WA 98195-1750, USA Received 1 June 2006; received in revised form 6 June 2007; accepted 7 June 2007 Available online 15 June 2007

Abstract We have used electrochemical processing to fabricate ordered arrays of metals and metal oxides on surfaces at densities exceeding 1012 cm−2 , on mm2 areas, and with typical feature sizes of 2–3 nm. This is achieved via masks obtained from naturally occurring proteins that assemble into two-dimensional crystals containing internal porous structure within each unit cell of the crystalline lattice. We have proven this process with bacterial cell surface proteins (S-layer proteins) from Deinococcus radiodurans and Sporosarcina ureae. Each of these S-layer proteins has unique lattice geometry and internal structure. Substrates are coated by adsorption from a dilute suspension of purified, stabilized protein extract. Electrochemical deposition proceeds through solvent accessible pores of the S-layer crystal to build surface structures with nanometer scale feature sizes and spacings precisely matching the geometry of the protein “mask”. Comparisons between the structure of the electrodeposited material through the protein mask and the protein surface topography suggest that the S-layers of D. radiodurans possess pores providing straight through-holes to the surface, whereas the S-layers of S. ureae presents a more tortuous pathway to the electrode surface. © 2007 Elsevier Ltd. All rights reserved. Keywords: Nanotechnology; Nanofabrication; Protein; Electrodeposition; Through-mask electroplating

1. Introduction Bacterial cell surface proteins (S-layers), first seen in lowresolution electron micrographs by Houwink [1] in 1953, are rapidly attracting much interest for nanotechnology applications [2,3]. Although many other proteins also assemble into twodimensional crystals, such as porins, chaperonins, and various integral membrane proteins [4], there are significant advantages of S-layer proteins. Typically, S-layer proteins are produced in reasonable abundance from bacterial sources without resorting to genetic engineering, many retain their crystalline state when purified, and they are unusually resistant to harsh chemicals and harmful enzymes [5]. Because of these properties, one of the most popular applications of S-layer proteins in nanotechnology is to employ them as nanoscale pattern-generating agents to build nanostructures that are impossible or difficult to synthesize via conventional methods. ∗

Corresponding author at: Chemical Engineering Department, University of Washington, Seattle, WA 98195-1750, USA. Tel.: +1 206 685 4815; fax: +1 206 543 3778. E-mail address: [email protected] (D.T. Schwartz). 1 ISE member. 0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.06.019

S-layer proteins have been exploited numerous times to organize nanoscale particles in solution by the reduction of precursor metal salts [6–9]. A parallel research effort has been underway that exploits S-layers in vapor-deposition processes to create nanostructured surfaces [10–13]. Our own research has demonstrated the compatibility of S-layer proteins with electrodeposition [14], a technique that can grow through complex masks in confined geometries [15,16]. With electrodeposition, solid material grows from the substrate out through ion-accessible solvated space within and between protein crystal subunits pre-adsorbed on the work surface. Moreover, S-layer proteins can themselves be patterned on surfaces by soft lithography [17] or by deep ultraviolet (UV) lithography [18], enabling hierarchical fabrication across wide length scales. In this publication, we provide detailed guidelines for using S-layer proteins in electrochemical fabrication processes, i.e., starting from a desired periodic structure to characterization of the final product. These guidelines arise from our experience working with three different organisms that produce three different S-layer proteins. In each case, the periodic geometry one seeks is defined, then a simple-to-grow microorganism possessing an S-layer protein matching the desired geometry is identified, the organism is grown, its S-layer harvested, puri-

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fied, and adsorbed to surfaces for subsequent electrodepositon. We demonstrate this process for the S-layers of Deinococcus radiodurans and Sporosarcina ureae, and we conclude this article with some outstanding unanswered questions about these exotic nanostructures. 2. Experimental 2.1. Cultivation of microorganisms D. radiodurans SARK (ATCC 35073) and S. ureae (ATCC 13881) were obtained as freeze-dried cultures and transferred to 5 mL of growth media: 0.5% peptone, 0.3% yeast extract, 0.1% glucose (TGY medium), for D. radiodurans and 3.0% tryptic soy broth (TSB medium), for S. ureae. These were grown overnight in rotary shakers at 30 ◦ C, and plated onto media solidified with 1.5% agar. Individual colonies were re-grown overnight at 30 ◦ C in the appropriate growth media. Glycerol was added to 24% (vol/vol) final concentration and 3 mL stocks were stored at −80 ◦ C in cryogenic tubes. For cultivation, a heated platinum wire loop was used to transfer a small amount of frozen cells to 5 mL of freshly prepared TGY or TSB media and cells were grown overnight at 30 ◦ C in rotary shakers. The overnight culture was used to inoculate 25 mL of media (by 50-fold dilution using sterile techniques) and cells were grown to early stationary phase. The resulting culture was used to inoculate 1 L of growth medium as above. Growth was monitored by measuring the optical density of the solution at 600 nm using a 96-well microplate reader with 200 ␮L of sample. Cells were harvested at early stationary phase and rinsed by centrifuging at 2000–3000 × g for 15 min and resuspended in deionized water three times. 2.2. S-layer protein purification S-layer proteins were purified by differential centrifugation. For D. radiodurans, cells were stripped of their S-layers by incubation in 50 mL 5 wt.% sodium dodecyl sulfate (SDS) for 2 h at 60 ◦ C. For S. ureae, cells were lysed by a French press operated at 20,000 psi. In both cases, the suspensions were centrifuged at low speed (2000–3000 × g) to remove denuded or unbroken cells, and the supernatant was transferred to fresh centrifuge tubes. S-layer proteins were recovered by centrifugation at 28,000 × g for 50 min. For D. radiodurans, the proteins were resuspended in 2 mL 5% SDS and the purification is complete. For S. ureae, protein pellets were resuspended in 30 mL 50 mM sodium phosphate pH 7.8, supplemented with 1% Triton X-100 and 1 mM MgCl2 (SPBM buffer), and incubated overnight at room temperature. On the next day, S-layer proteins were pelleted by centrifugation at 28,000 × g for 50 min, resuspended in 30 mL of SPBM buffer and the process was repeated three times. Finally, lysozyme (Sigma) was added to 100 ␮g mL−1 final concentration and the suspension incubated overnight at 37 ◦ C in a rotary shaker. Proteins were pelleted and washed in deionized water with 1% Triton X-100 three times as above. S-layer proteins from D. radiodurans were stored at room temperature. S-layer proteins from S. ureae were stored at 4 ◦ C. Both protein products were stored as stocks at 1 mg mL−1 con-

centration based on the Bradford assay [19] using bovine serum albumin (BSA) as the standard. 2.3. Templated electrodeposition Stock protein suspensions were adsorbed for about 10 s onto platinum-coated gold TEM grids (see Section 2.4) and then the surface was rinsed with deionized water. This short contact time provided 20–40% protein coverage on the surface, comprised of randomly arranged protein crystal fragments approximately 1–2 ␮ in size, with occasional folds and tears. It was noted that S-layers of S. ureae were easily rinsed off electrode surfaces and so rinsing was performed more carefully by repeated immersion rather than by squirting deionized water over the surface with a squirt bottle. Electrodeposition of cuprous oxide was performed by room temperature reduction from an aqueous electrolyte of 0.4 M CuSO4 , 3 M lactic acid, and pH adjusted to 9.0 via addition of NaOH [20]. A three-electrode cell was used under quiescent conditions. Electrodeposition proceeded at −450 mV versus SCE (saturated calomel electrode) for 15 min. 2.4. Imaging and characterization Atomic force microscopy (AFM) was performed on a Nanoscope III AFM in Tapping Mode® using ∼300 kHz aluminum-coated silicon cantilevers with 10 nm radius tips (molecular imaging) at a scan rate of 1 Hz with an image field of 750 nm. AFM samples were prepared by adsorption of protein onto freshly cleaved mica and washing as described for templated electrodeposition. Transmission electron microscopy (TEM) was performed on a Philips 420 TEM at 120 kV accelerating voltage with no objective aperture. Samples were prepared by coating 400-mesh gold TEM grids with 2 nm platinum films prepared by argonion sputtering onto surfactant-coated glass slides [21]. Such platinum-coated TEM grids were used as the working electrodes in electrodeposition experiments and no further sample preparation was required after electrodeposition. The effective camera length for electron diffraction was calibrated with an aluminum foil standard. 3. Results and discussion Table 1 compiles the most relevant information for S-layer based nanofabrication. It draws from an extensive S-layer protein research literature, as well as several existing tabulations of S-layer carrying organisms [22,23]. The S-layer proteins shown in Table 1 were selected for the purpose of demonstrating the variety of systems available, not only in terms of structure, but also in terms of difficulty or ease with which the organisms are grown and the S-layer proteins are purified. The compilation is not intended to be comprehensive. With respect to protein stability, it is important to note that these studies were generally performed in solution with common protein denaturing agents. One might expect a different behavior with proteins adsorbed on a surface in the presence

Table 1 Examples of S-layer proteins highlighting the variety of structures potentially available Fabricated structure Densitya

(# × 1012 cm−2 )

References

Species and hazard class

(nm)

Geobacillus stearothermophilus NRS 2004/3a Bacillus coagulans E38-66 Lobomonas piriformis Chlamydomonas reinhardi Aquaspirillum putridiconchlyum Sulfolobus shibatae Acidianus brierleyi Aeromonas salmonicida Desulfurococcus mobilis Sporosarcina ureae Deinococcus radiodurans SARK Chlamydia trachomatis TE55 Caulobacter crescentus 15NY106

p1

10 × 8, 81◦

2.8

2.5

[25]

p1 p2 p2 p2 p3 p3 p4 p4 p4 p6 p6 p6

9 × 7, 80◦ 24 × 16, 109◦ 29 × 24, 80◦ 12 × 8, 73◦ 21 19 11 18 13 18 18 24

3.0 6.5 2.5 1.8 3.8 4.3 2.0 5.4 1.7 3.4 3.2 3.8

4.3 0.8 0.3 3.4 0.3 1.0 0.8 0.3 0.8 1.1 2.6 0.4

[29] [32] [34] [36] [38] [41] [43] [45] [47] [49] [53] [57]

a

Protein details BSL#

Sourceb

Growthc

Purificationd

Stabilitye

1

F. Hollaus, Austria [26]

T+ O2 − [27]

X RC [28]

D E [26]

1 1 1 1 1 1 2 1 1 1 2 1

F. Hollaus, Austria CCAP 45/1 ATCC 18798 ATCC 15279 DSM 5389 DSM 1651 C. Michel, France [44] DSM 2161 ATCC 13881 ATCC 35073 F.-F. T’ang, China [54] J. Poindexter, NY [58]

T+ [30] ill [32] ill [35] Common [37] T+ ac [39] T+ S0 ac [41] T− [43] T+ O2 − S0 [46] Common [47] Common [50] inf H DC [55,56] Common [57]

L X RC [30,31] DC [33] L DC [34] DC [37] L D DC [40] L D DC [41] L DC [44] L D DC [45] L DC D E [47] D DC [51] D DC E [53] DC [57]

? ? ? pH sol M+ [37] ? D [42] ? ? pH M+ D E [48] pH M+ D E [52] D E [56] pH M+ [57]

Minimum feature size and bit density is estimated based on the protein structure reconstructions cited in the reference. (ATCC) Amer. Type Cult. Collec.; (DSM) Germ. Collec. of Microorg. and Cell Cult.; (CCAP) Cult. Centre of Algae and Protozoa (UK). c (T+ ) High T (>37 ◦ C); (T− ) low T (<30 ◦ C); (O − ) limited to no oxygen; (ill) light; (S0 ) elemental sulfur required; (ac) acidic pH; (inf) infection of tissue; (H) homogenization of infectant; (DC) differential 2 centrifugation; (common) 30–37 ◦ C, open shake flask. d (L) lysis; (D) detergent treatment; (X) chaotropic disruption; (RC) recrystallization; (DC) differential centrifugation; (E) enzyme treatment. e (pH) pH; (sol) solvent; (M+ ) solvated cationic content; (D) detergent; (E) enzymes; (?) no significant study known to us. b

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Unit cell geometry (nm × nm, β angle)

Host organism details Sizea

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of the strong electrolytes typical in electrochemical fabrication processes. From Table 1, we initially selected three different systems for electrochemical nanofabrication, the S-layers from D. radiodurans, S. ureae, and Desulfurococcus mobilis. We have successfully fabricated cuprous oxide arrays through the Slayers of both D. radiodurans and S. ureae, which are presented in this article, but have not yet been successful with the S-layers of D. mobilis. As Table 1 summarizes, the microorganisms D. radiodurans and S. ureae are both cultured in very simple environments, whereas D. mobilis requires anaerobic conditions, extremely high temperatures, and elemental sulfur for growth, a cultivation condition which is still not very well understood biochemically [24]. We have yet to obtain our own S-layers from D. mobilis because we have not yet successfully grown this organism. Fig. 1 depicts the growth curves of the two S-layer producing organisms used in this study: D. radiodurans var. SARK and S. ureae. Such plots typically show the change in the optical density (turbidity) of a culture with time at a wavelength where neither medium nor biological components absorb light (usually 600 nm). To facilitate estimation of S-layer protein recovery, the plots of Fig. 1 were further calibrated to dry cell weight. Note that the presence of elemental sulfur particles complicates the construction of a growth curve for an organism like D. mobilis using this method. Inspection of the growth curves reveals the transition from the exponential phase of growth (where cells grow at their maximal possible rate) to the stationary phase, at which point all nutrients in a batch culture have been exhausted and there is no net cell growth. To maximize the recovery yield of S-layer proteins, the optimal time to harvest is in early stationary phase. After cells have been harvested, the next step is to purify Slayer proteins. There are many different approaches to this task,

Fig. 1. Growth curves of (A) Deinococcus radiodurans, and (B) Sporosarcina ureae. The transition from logarithmic phase to stationary phase is indicated, representing the opportune time to harvest S-layer proteins. Growth was monitored by measuring the optical density of the solution at λ = 600 nm using a 96-well microwell plate reader with 200 ␮L aliquots of culture. Dry cell mass was calibrated to the optical density by weighing out cell samples after three cycles of centrifugation and washing followed by lyophilization for 4–5 h.

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electropherosis (SDS-PAGE) of protein samples for the organisms used in this study. Labels denote whether the sample analyzed corresponds to whole cell (WC) or purified S-layer protein (SL). Sources are indicated. Lane (M) contains marker proteins for calibration of molecular weight. Arrows shows the position of full-length S-layer proteins, approximately 105,000 g mol−1 for D. radiodurans and 116,000 g mol−1 for S. ureae. Lower molecular mass species are likely to be cleaved fragments of the intact protein, rather than unrelated protein contaminants.

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as shown in Table 1. In the case of D. radiodurans, a simple wash in sodium dodecyl sulfate (SDS) at high temperature (60–80 ◦ C) causes large crystalline patches of S-layer proteins to slough off from the cells intact [14,59,60]. The resulting proteins can be further purified by differential centrifugation, which relies on the fact that cells are heavier than S-layer crystals, which are themselves heavier than individual proteins present in the suspension. In the case of S. ureae, the protein crystals remain attached to cell wall components, which we digest by treatment with Triton X-100 and lysozyme to ensure that electrodeposition will not be blocked. While this separation method allows convenient access to S-layer proteins, it is not universally applicable or even ideal in certain cases. Some limitations include the size of the crystalline fragments that can be obtained (the size depends on the originating organism, often 1–2 ␮m), the occurrence of overlapping fragments, and the possible adsorption of the protein sheets with different faces toward the electrolyte. Gel electrophoresis provides evidence for the purity of the resulting S-layer proteins [61]. In this technique, proteins are fractioned on the basis of their molecular masses. Fig. 2 shows such gels for the two S-layer proteins used in this study. The marker lane (M) contains purified proteins of known molecular mass that allows one to calculate the mass of the target protein by interpolation. The whole cell lane (WC) shows the total proteins contained in the cell prior to purification. The Slayer lane (SL) shows S-layer proteins after the purification steps described above have been performed. Note that the (SL) lanes not only contain a product corresponding to the expected molecular mass of purified S-layer proteins (arrow) but additional “lighter” products exhibiting higher electrophoretic mobility. These polypeptides likely correspond to the processing of fulllength S-layer proteins by a highly specific proteolytic enzyme endogenous to the organisms [62]. We have not found these to be detrimental for creating high quality S-layer masks for electrochemical nanofabrication. Once the purified proteins are suspended in stabilizing media, often a 1–2 wt.% solution of detergent, such as sodium dodecyl sulfate (SDS), Triton X-100, or Tween 20, it is possible to coat surfaces with the proteins for fabrication. Fig. 3 shows examples of how S-layer proteins from D. radiodurans (A) and S. ureae (B) appear by atomic force microscopy (AFM) upon adsorption onto mica. The Fast Fourier transform insets clearly show the expected symmetry: hexagonal for the D. radiodurans S-layer and square for the S. ureae S-layer. Additional space group symmetries are available by making use of other S-layer-producing organisms (Table 1). For the S-layers of D. radiodurans, the AFM image clearly shows hexagonally arranged “pores” through the protein. Electron density mapping has confirmed that the pores traverse the protein [63]. Internal pore structure is not clearly evidenced in AFM results on S. ureae S-layers. Electron density mapping of this protein has revealed the tortuous and complex structure of this protein [47]. Major benefits of electrochemical processing for growth of nanostructures include (1) growth can proceed through tortuous pathways to create three-dimensional structures so long as they are ion-accessible and (2) the deposited structure is dictated by the crystallinity of the proteins at the elec-

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Fig. 3. Topographic images of S-layer proteins on freshly cleaved mica obtained by Tapping Mode® atomic force microscopy (AFM) using aluminum-coated 10 nm radius AFM probes from (A) D. radiodurans, and (B) S. ureae. Insets show Fast Fourier transforms of the image revealing the six-fold and four-fold symmetry, respectively.

trode surface. Because S-layers fully cover their host organisms, every S-layer protein is likely to have continuous ion-accessible channels for electrodeposition. We have performed systematic investigations on the nanostructure observed after electrodeposition of cuprous oxide. Fig. 4 shows a transmission electron microscope study of electrodeposited cuprous oxide, a material that provides high quality deposits by nucleating with sufficient density to template a significant fraction of the protein mask. Cu2 O was clearly identified in electron diffraction patterns. The electron diffraction patterns obtained from the patterned regions indicate that the deposit is

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with multilayer S-layer protein crystals does not impede electrodeposition of nanostructures through the protein “mask” [14]. In the case of electrodeposition through S-layers of D. radiodurans, we observe a structure, which agrees with the topography observed in AFM images, i.e., the resulting deposit is a periodic array of cuprous oxide nanocrystals and the protein mask may be described as a simple pattern of through-holes. For the S-layers of S. ureae we observe a significantly more complex deposit with structure finer than the periodicity of the protein array, implying a significant three-dimensional component to the structure. Not much is presently known about this deposited structure, though tentatively, it appears to be composed of a mesh of interconnected nanocrystalline cuprous oxide. 4. Conclusions and implications This article expands our previous work on templating through S-layer protein crystals by electrodeposition, and provides a road map (Table 1) for creating other nanofabricated structures with S-layers by electrodeposition. As Table 1 shows, some Slayer proteins have been evaluated for their stability in selected electrolytes. Interestingly, we have recently shown that the twodimensional structure of an S-layer can be disrupted in certain electrolytes well before there is damage to the underlying protein [68]. For example, we have found the S-layers of D. radiodurans to lose their crystallinity in the presence of a typical copperplating bath of pH 0, although gel electrophoresis demonstrated protein stability in this bath. We performed bath engineering by raising the pH to 3 while maintaining ionic strength with magnesium ions, which allowed the S-layers to maintain their crystallinity and nanofabrication was successful. Finally, we hope to achieve a full 3D reconstruction of the deposited material by electron tomography and compare it to the structure of the proteins themselves. This will reveal which portions of the protein are not accessible for electrodeposition. Answering these questions is central to expanding the use of S-layer proteins in electrochemical fabrication processes. Acknowledgements Fig. 4. Transmission electron micrographs of cuprous oxide electrodeposited on platinum-coated specimen grids previously adsorbed with S-layer proteins from (A) D. radiodurans, and (B) S. ureae. Upper left insets are Fast Fourier transform filtered images, and upper right insets represent electron diffraction patterns of the patterned regions, which agree with simulated powder diffraction patterns (superimposed) of crystalline cuprous oxide. Pattern geometry and spacings match those seen in AFM images to within instrumental accuracy.

in the form of very small crystallites, resulting in the speckled contrast as seen in Fig. 4 as a result of diffraction contrast. This would not be the case if the image resulted from “negative staining” [64–67] when the proteins are immersed in the electrolyte, which would otherwise result in diffuse electron diffraction patterns. While these studies used surfaces partially coated with protein crystals so that nucleation and growth on protein-free regions could be observed under identical processing conditions, we have previously shown that 100% surface coverage

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