- Email: [email protected]
Micro-“factory” for self-assembled peptide nanostructures
Microelectronic Engineering 88 (2011) 1685–1688
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Micro-‘‘factory’’ for self-assembled peptide nanostructures Jaime Castillo-León a,⇑, Romén Rodriguez-Trujillo a, Sebastian Gauthier a, Alexander C.Ø. Jensen b, Winnie E. Svendsen a a b
Technical University of Denmark, Lyngby 2800, Denmark Copenhagen University, Copenhagen 2100, Denmark
a r t i c l e
i n f o
Article history: Available online 21 December 2010 Keywords: Microfluidics Self-assembled peptides Nanostructures Peptide tube Peptide nanoparticle On-chip fabrication
a b s t r a c t This study describes an integrated micro ‘‘factory’’ for the preparation of biological self-assembled peptide nanotubes and nanoparticles on a polymer chip, yielding controlled growth conditions. Self-assembled peptides constitute attractive building blocks for the fabrication of biological nanostructures due to the mild conditions of their synthesis process. This biological material can form nanostructures in a rapid way and the synthesis method is less expensive as compared to that of carbon nanotubes or silicon nanowires. The present article thus reports on the on-chip fabrication of self-assembled peptide nanostructures by means of a microfluidic device that is able to resist the harsh conditions imposed by the solvent used during the nanostructure synthesis. This on-chip fabrication was found to be simple, rapid, and convenient. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Self-assembled peptide building blocks are very attractive biological materials for bionanotechnology applications due to their low cost, chemical flexibility and biocompatibility [1]. Moreover, self-assembled peptides constitute an attractive route for the synthesis of functional nanomaterials. Diphenylalanine (FF) is an aromatic dipeptide capable of rapidly self-assembling into hollow tubular nanostructures under mild conditions [2]. The peptide nanotubes formed by this building block have demonstrated unique mechanical, thermal and chemical properties [3,4]. These biological nanotubes have been extensively used in a variety of bionanotechnology applications such as electrochemical biosensors, scaffolds for the fabrication of metallic nanowires, coaxial nanocables, and the development of photoluminiscent nanotubes, among others [1,5]. Their electrical and structural properties have been recently studied [6,7]. An aromatic dipeptide analogue, i.e., tertbutoxycarbonyl–Phe– Phe–OH (Boc–Phe–Phe–OH), can self-assemble into two types of nanostructures, i.e., nanotubes and nanoparticles, under varying synthesis conditions [8]. A big challenge in peptide-based nanofabrication is to control the synthesis and the resulting size of these biological nanostructures. The synthesis of FF nanotubes was first presented by Reches and Gazit [2] and their method was later
modified for the fabrication of Boc–Phe–Phe–OH nanoparticles [8]. This facile fabrication method results in the immediate formation of peptide nanoparticle agglomerates with sizes ranging from a few nanometers to several micrometers. A more controlled synthesis method would facilitate the integration of this promising biomaterial in bionanotechnological processes such as the development of biosensing devices or drug delivery, where size control is an essential feature [9]. The present communication reports on the fabrication and performance of a microfluidic chip for the synthesis of selfassembled structures, tubes and particles. Due to their dimensions, the flow in microfluidic channels is laminar, and two or more liquids flowing in contact with each other in a laminar regime mix only by diffusion [10]. These properties, characteristic to microfluidic devices, have previously been utilized in biochemical assays, separation, micro-fabrication, kinetic analysis, biofilm formation monitoring as well as in protein interaction [10,11]. The presence of a laminar flow in the presented microfluidic device should render it possible to obtain a precise synthesis of biological particles on an aqueous–organic interface. This way, the obtained nanostructures would display a more uniform shape and size.
2. Fabrication of the microfluidic device
⇑ Corresponding author. Tel.: +45 45256837. E-mail address: [email protected] (J. Castillo-León). 0167-9317/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2010.12.023
The microfluidic chip prepared during this study was composed of two polydimethylsiloxane (PDMS) layers, where the bottom layer was fabricated with a PDMS stamping technique.
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J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
Fig. 1. An illustration of the designed microfluidic chip. Three inlets were used for injecting the structure precursors. The inset shows a simulation of the mixing of different solutions inside the merging channel, where the nanotubes are expected to be formed.
A silicon stamp was created using standard cleanroom fabrication techniques (photolithography and advanced silicon etching) to create 100-lm deep channels. The PDMS was then poured on the silicon wafer in an ad hoc container. After the curing process, the PDMS layer was carefully removed. The second (top) layer was fabricated by injection molding. The mold was built by assembling two micromachined poly(methyl methacrylate) (PMMA) layers and tightening them with screws and nuts. The sealing of the two layers was then ensured by applying a mechanical pressure. Subsequently, the PDMS was squeezed between the two PMMA layers endowed with holes to allow the connections. Fig. 1 presents a schematic of the microfluidic chip. As can be seen, the chip had three inlets for injecting reaction products, 1–3, and one outlet, 4, for collecting the formed nanotubes. Inlet 1 was used to inject double-distilled water, and inlet 2 was used to inject 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) while inlet 3 was used to inject the lyophilized peptide dissolved in HFP. The three microchannels merged with each other thus creating a reaction zone between the merging point and the outlet.
4. Results 4.1. Microfluidic device fabrication The fabricated chip must fulfill two requirements: it should be transparent to allow the optical observation of the synthesis process, and the material used in the fabrication of the chip should resist the conditions imposed by the presence of HFP, a fluorinated alcohol utilized to dissolve the lyophilized peptide. It was found during a previous investigation that HFP could dissolve certain polymers at room temperature [12]. Consequently, a solubility test of several polymers commonly used in micro-fabrication was performed in an HFP solution. Square samples, 1 1 cm2, of five polymers were immersed and maintained overnight in an HFP solution at room temperature. The solubility of the specimens was determined the next day, and the obtained results are presented in Supplementary Table 1. Only PDMS and cyclic olefin copolymer (TOPAS) could resist dissolution by the alcohol, whereas the other three polymers became completely or partially dissolved. Finally, PDMS was chosen for the chip fabrication due to its resistance to HFP, as well as its simple, rapid and low-cost fabrication process.
3. Fabrication of self-assembled peptide nanostructures 4.2. On-chip fabrication of self-assembled nanostructures The traditional synthesis of FF tubes and Boc–Phe–Phe–OH particles starts with the preparation of a peptide stock solution: the lyophilized peptides are dissolved in HFP to a final concentration of 100 mg/mL at room temperature. FF tubes were then prepared by dissolving aliquots of the peptide stock solution in water to a final concentration of 2 mg/mL. Boc–Phe–Phe–OH particles were fabricated by dissolving aliquots of the peptide stock solution in 50% ethanol to a final concentration of 50 mg/mL. For the on-chip fabrication of FF and Boc–Phe–Phe–OH structures, the chip was first assembled and cleaned with MilliQ water. Subsequently, pure HFP was flushed through the alcohol inlet to remove any water from the HFP channel. The extra inlet for HFP injection was necessary in order to establish a laminar flow between the alcohol and the water. For the preparation of the BOC particles, a similar procedure was followed, however with a 50% solution of ethanol instead of water.
The formation of a biphasic laminar flow was confirmed and visualized by the injection of a dye solution into the microchannels. In the mixing channel, where the two liquids merged, it was observe how the blue dye and uncolored water became separated by the interface. The laminar flow was consistently obtained, independently of the varying flow rates used to inject the precursor solutions. This feature was a key parameter in order for the self-assembled structures to form exactly at the interface between the aqueous and the organic phase created when the water and the peptide stock solution met inside the main channel. 4.3. Evaluation of the on-chip synthesized self-assembled nanostructures The on-chip synthesized structures were collected and visualized by scanning electron microscopy (SEM) and transmission
J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
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Fig. 2. SEM images of FF tubes fabricated according to the traditional method, left image, and counterparts synthesized on-chip, right image.
Fig. 3. SEM images of the Boc–Phe–Phe–OH particles fabricated according to the traditional method, left, and counterparts synthesized on-chip, right image.
Fig. 4. A TEM image of the on-chip synthesized Boc–Phe–Phe–OH particles.
as seen in the figures, the on-chip synthesized structures displayed more uniform shapes and sizes. The structures were visible during the synthesis process inside the chip. The size of the FF tubes was dependent of the flow rate chosen as can be seen in Supplementary Figure S1. Additionally the SEM images in Fig. 2 show that the obtained structures are in fact tubes and not wires, as evident by the presence of an internal cavity in Fig. 2, right. A more detailed image of the cavity of the on-chip fabricated tube is displayed in more detail in Supplementary Figure S2. These results are consistent with our previous work involving the structural characterization of this type of structures using Electrostatic Force Microscopy [7]. In Fig. 3 a SEM image of synthesized nanoparticles is shown. In the left image the traditional synthesis is used while in the right image the particles are synthesized on-chip. As can be seen, the on-chip synthesized particles displayed more uniform shapes and sizes. In order to evaluate if the synthesized nanoparticles were solid or hollow, the structures were further analyzed by TEM. Fig. 4 displays a TEM image showing that the synthesized nanoparticles were in fact solid spheres. The black center of the nanoparticles in the TEM images indicated that the electrons were dispersed through the particle by the molecules inside. In addition, the TEM image confirmed the uniform shape of the obtained structures. 5. Conclusions
electron microscopy (TEM). Figs. 2 and 3 show a comparison between the structures fabricated using the traditional method, left image, and structures synthesized on-chip, right image. In general
A microfluidic PDMS chip for the synthesis of self-assembled structures was fabricated. The device was resistant to the HFP
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J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
alcohol which is known to attack several polymers. The on-chip fabricated structures displayed a more uniform size and shape as compared to counterparts prepared according to the traditional method. Through SEM and TEM analysis was possible to confirm that the on-chip fabricated structures were hollow tubes and solid particles. The presented microfluidic device offers the possibility of future integration of manipulation elements such as electrodes for dielectrophoresis or pinch flow segments for size separation, as well as the inclusion of new flows carrying compounds for the functionalization of the fabricated structures.
Acknowledgments The European Community (BeNatural/NMP4-CT-2006-033256) and the Danish Agency for Science Technology and Innovation (FTP 271-08-0968) are gratefully acknowledged for financial support. The authors also thank Dr. Casper Clausen for his help with the TEM analysis.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mee.2010.12.023. References [1] X.H. Yan, P.L. Zhu, J.B. Li, Chem. Soc. Rev. 39 (2010) 1877–1890. [2] M. Reches, E. Gazit, Sciences 300 (2003) 625–627. [3] N. Kol, L. Adler-Abramovich, D. Barlam, R.Z. Shneck, E. Gazit, I. Rousso, Nano Lett. 5 (2005) 1343–1346. [4] J. Ryu, C.B. Park, Biotechnol. Bioeng. 105 (2010) 221–230. [5] S. Scanlon, A. Aggeli, Nano Today 3 (2008) 22–30. [6] J. Castillo, S. Tanzi, M. Dimaki, W.E. Svendsen, Electrophoresis 29 (2008) 5026– 5032. [7] C.H. Clausen, J. Jensen, J. Castillo, M. Dimaki, W.E. Svendsen, Nano Lett. 8 (2008) 4066–4069. [8] L. Adler-Abramovich, E. Gazit, J. Pept. Sci. 14 (2008) 217–223. [9] A. MaHam, Z. Tang, H. Wu, J. Wang, Y. Lin, Small 5 (2009) 1706–1721. [10] B.H. Weigl, P. Yager, Science 283 (1999) 346–347. [11] S. Marre, K.F. Jensen, Chem. Soc. Rev. 39 (2010) 1183–1202. [12] T.H. Mourey, T.G. Bryan, J. Chromatogr. A 964 (2002) 169–178.
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Micro-‘‘factory’’ for self-assembled peptide nanostructures Jaime Castillo-León a,⇑, Romén Rodriguez-Trujillo a, Sebastian Gauthier a, Alexander C.Ø. Jensen b, Winnie E. Svendsen a a b
Technical University of Denmark, Lyngby 2800, Denmark Copenhagen University, Copenhagen 2100, Denmark
a r t i c l e
i n f o
Article history: Available online 21 December 2010 Keywords: Microfluidics Self-assembled peptides Nanostructures Peptide tube Peptide nanoparticle On-chip fabrication
a b s t r a c t This study describes an integrated micro ‘‘factory’’ for the preparation of biological self-assembled peptide nanotubes and nanoparticles on a polymer chip, yielding controlled growth conditions. Self-assembled peptides constitute attractive building blocks for the fabrication of biological nanostructures due to the mild conditions of their synthesis process. This biological material can form nanostructures in a rapid way and the synthesis method is less expensive as compared to that of carbon nanotubes or silicon nanowires. The present article thus reports on the on-chip fabrication of self-assembled peptide nanostructures by means of a microfluidic device that is able to resist the harsh conditions imposed by the solvent used during the nanostructure synthesis. This on-chip fabrication was found to be simple, rapid, and convenient. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Self-assembled peptide building blocks are very attractive biological materials for bionanotechnology applications due to their low cost, chemical flexibility and biocompatibility [1]. Moreover, self-assembled peptides constitute an attractive route for the synthesis of functional nanomaterials. Diphenylalanine (FF) is an aromatic dipeptide capable of rapidly self-assembling into hollow tubular nanostructures under mild conditions [2]. The peptide nanotubes formed by this building block have demonstrated unique mechanical, thermal and chemical properties [3,4]. These biological nanotubes have been extensively used in a variety of bionanotechnology applications such as electrochemical biosensors, scaffolds for the fabrication of metallic nanowires, coaxial nanocables, and the development of photoluminiscent nanotubes, among others [1,5]. Their electrical and structural properties have been recently studied [6,7]. An aromatic dipeptide analogue, i.e., tertbutoxycarbonyl–Phe– Phe–OH (Boc–Phe–Phe–OH), can self-assemble into two types of nanostructures, i.e., nanotubes and nanoparticles, under varying synthesis conditions [8]. A big challenge in peptide-based nanofabrication is to control the synthesis and the resulting size of these biological nanostructures. The synthesis of FF nanotubes was first presented by Reches and Gazit [2] and their method was later
modified for the fabrication of Boc–Phe–Phe–OH nanoparticles [8]. This facile fabrication method results in the immediate formation of peptide nanoparticle agglomerates with sizes ranging from a few nanometers to several micrometers. A more controlled synthesis method would facilitate the integration of this promising biomaterial in bionanotechnological processes such as the development of biosensing devices or drug delivery, where size control is an essential feature [9]. The present communication reports on the fabrication and performance of a microfluidic chip for the synthesis of selfassembled structures, tubes and particles. Due to their dimensions, the flow in microfluidic channels is laminar, and two or more liquids flowing in contact with each other in a laminar regime mix only by diffusion [10]. These properties, characteristic to microfluidic devices, have previously been utilized in biochemical assays, separation, micro-fabrication, kinetic analysis, biofilm formation monitoring as well as in protein interaction [10,11]. The presence of a laminar flow in the presented microfluidic device should render it possible to obtain a precise synthesis of biological particles on an aqueous–organic interface. This way, the obtained nanostructures would display a more uniform shape and size.
2. Fabrication of the microfluidic device
⇑ Corresponding author. Tel.: +45 45256837. E-mail address: [email protected] (J. Castillo-León). 0167-9317/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2010.12.023
The microfluidic chip prepared during this study was composed of two polydimethylsiloxane (PDMS) layers, where the bottom layer was fabricated with a PDMS stamping technique.
1686
J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
Fig. 1. An illustration of the designed microfluidic chip. Three inlets were used for injecting the structure precursors. The inset shows a simulation of the mixing of different solutions inside the merging channel, where the nanotubes are expected to be formed.
A silicon stamp was created using standard cleanroom fabrication techniques (photolithography and advanced silicon etching) to create 100-lm deep channels. The PDMS was then poured on the silicon wafer in an ad hoc container. After the curing process, the PDMS layer was carefully removed. The second (top) layer was fabricated by injection molding. The mold was built by assembling two micromachined poly(methyl methacrylate) (PMMA) layers and tightening them with screws and nuts. The sealing of the two layers was then ensured by applying a mechanical pressure. Subsequently, the PDMS was squeezed between the two PMMA layers endowed with holes to allow the connections. Fig. 1 presents a schematic of the microfluidic chip. As can be seen, the chip had three inlets for injecting reaction products, 1–3, and one outlet, 4, for collecting the formed nanotubes. Inlet 1 was used to inject double-distilled water, and inlet 2 was used to inject 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) while inlet 3 was used to inject the lyophilized peptide dissolved in HFP. The three microchannels merged with each other thus creating a reaction zone between the merging point and the outlet.
4. Results 4.1. Microfluidic device fabrication The fabricated chip must fulfill two requirements: it should be transparent to allow the optical observation of the synthesis process, and the material used in the fabrication of the chip should resist the conditions imposed by the presence of HFP, a fluorinated alcohol utilized to dissolve the lyophilized peptide. It was found during a previous investigation that HFP could dissolve certain polymers at room temperature [12]. Consequently, a solubility test of several polymers commonly used in micro-fabrication was performed in an HFP solution. Square samples, 1 1 cm2, of five polymers were immersed and maintained overnight in an HFP solution at room temperature. The solubility of the specimens was determined the next day, and the obtained results are presented in Supplementary Table 1. Only PDMS and cyclic olefin copolymer (TOPAS) could resist dissolution by the alcohol, whereas the other three polymers became completely or partially dissolved. Finally, PDMS was chosen for the chip fabrication due to its resistance to HFP, as well as its simple, rapid and low-cost fabrication process.
3. Fabrication of self-assembled peptide nanostructures 4.2. On-chip fabrication of self-assembled nanostructures The traditional synthesis of FF tubes and Boc–Phe–Phe–OH particles starts with the preparation of a peptide stock solution: the lyophilized peptides are dissolved in HFP to a final concentration of 100 mg/mL at room temperature. FF tubes were then prepared by dissolving aliquots of the peptide stock solution in water to a final concentration of 2 mg/mL. Boc–Phe–Phe–OH particles were fabricated by dissolving aliquots of the peptide stock solution in 50% ethanol to a final concentration of 50 mg/mL. For the on-chip fabrication of FF and Boc–Phe–Phe–OH structures, the chip was first assembled and cleaned with MilliQ water. Subsequently, pure HFP was flushed through the alcohol inlet to remove any water from the HFP channel. The extra inlet for HFP injection was necessary in order to establish a laminar flow between the alcohol and the water. For the preparation of the BOC particles, a similar procedure was followed, however with a 50% solution of ethanol instead of water.
The formation of a biphasic laminar flow was confirmed and visualized by the injection of a dye solution into the microchannels. In the mixing channel, where the two liquids merged, it was observe how the blue dye and uncolored water became separated by the interface. The laminar flow was consistently obtained, independently of the varying flow rates used to inject the precursor solutions. This feature was a key parameter in order for the self-assembled structures to form exactly at the interface between the aqueous and the organic phase created when the water and the peptide stock solution met inside the main channel. 4.3. Evaluation of the on-chip synthesized self-assembled nanostructures The on-chip synthesized structures were collected and visualized by scanning electron microscopy (SEM) and transmission
J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
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Fig. 2. SEM images of FF tubes fabricated according to the traditional method, left image, and counterparts synthesized on-chip, right image.
Fig. 3. SEM images of the Boc–Phe–Phe–OH particles fabricated according to the traditional method, left, and counterparts synthesized on-chip, right image.
Fig. 4. A TEM image of the on-chip synthesized Boc–Phe–Phe–OH particles.
as seen in the figures, the on-chip synthesized structures displayed more uniform shapes and sizes. The structures were visible during the synthesis process inside the chip. The size of the FF tubes was dependent of the flow rate chosen as can be seen in Supplementary Figure S1. Additionally the SEM images in Fig. 2 show that the obtained structures are in fact tubes and not wires, as evident by the presence of an internal cavity in Fig. 2, right. A more detailed image of the cavity of the on-chip fabricated tube is displayed in more detail in Supplementary Figure S2. These results are consistent with our previous work involving the structural characterization of this type of structures using Electrostatic Force Microscopy [7]. In Fig. 3 a SEM image of synthesized nanoparticles is shown. In the left image the traditional synthesis is used while in the right image the particles are synthesized on-chip. As can be seen, the on-chip synthesized particles displayed more uniform shapes and sizes. In order to evaluate if the synthesized nanoparticles were solid or hollow, the structures were further analyzed by TEM. Fig. 4 displays a TEM image showing that the synthesized nanoparticles were in fact solid spheres. The black center of the nanoparticles in the TEM images indicated that the electrons were dispersed through the particle by the molecules inside. In addition, the TEM image confirmed the uniform shape of the obtained structures. 5. Conclusions
electron microscopy (TEM). Figs. 2 and 3 show a comparison between the structures fabricated using the traditional method, left image, and structures synthesized on-chip, right image. In general
A microfluidic PDMS chip for the synthesis of self-assembled structures was fabricated. The device was resistant to the HFP
1688
J. Castillo-León et al. / Microelectronic Engineering 88 (2011) 1685–1688
alcohol which is known to attack several polymers. The on-chip fabricated structures displayed a more uniform size and shape as compared to counterparts prepared according to the traditional method. Through SEM and TEM analysis was possible to confirm that the on-chip fabricated structures were hollow tubes and solid particles. The presented microfluidic device offers the possibility of future integration of manipulation elements such as electrodes for dielectrophoresis or pinch flow segments for size separation, as well as the inclusion of new flows carrying compounds for the functionalization of the fabricated structures.
Acknowledgments The European Community (BeNatural/NMP4-CT-2006-033256) and the Danish Agency for Science Technology and Innovation (FTP 271-08-0968) are gratefully acknowledged for financial support. The authors also thank Dr. Casper Clausen for his help with the TEM analysis.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mee.2010.12.023. References [1] X.H. Yan, P.L. Zhu, J.B. Li, Chem. Soc. Rev. 39 (2010) 1877–1890. [2] M. Reches, E. Gazit, Sciences 300 (2003) 625–627. [3] N. Kol, L. Adler-Abramovich, D. Barlam, R.Z. Shneck, E. Gazit, I. Rousso, Nano Lett. 5 (2005) 1343–1346. [4] J. Ryu, C.B. Park, Biotechnol. Bioeng. 105 (2010) 221–230. [5] S. Scanlon, A. Aggeli, Nano Today 3 (2008) 22–30. [6] J. Castillo, S. Tanzi, M. Dimaki, W.E. Svendsen, Electrophoresis 29 (2008) 5026– 5032. [7] C.H. Clausen, J. Jensen, J. Castillo, M. Dimaki, W.E. Svendsen, Nano Lett. 8 (2008) 4066–4069. [8] L. Adler-Abramovich, E. Gazit, J. Pept. Sci. 14 (2008) 217–223. [9] A. MaHam, Z. Tang, H. Wu, J. Wang, Y. Lin, Small 5 (2009) 1706–1721. [10] B.H. Weigl, P. Yager, Science 283 (1999) 346–347. [11] S. Marre, K.F. Jensen, Chem. Soc. Rev. 39 (2010) 1183–1202. [12] T.H. Mourey, T.G. Bryan, J. Chromatogr. A 964 (2002) 169–178.