Invited Paper
Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds
Daniel Aili,† Hsu Shu Han,† Karin Enander,† Lars Baltzer,‡ and Bo Liedberg†*
†
Division of Sensor Science and Molecular Physics, Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden, and ‡Division of Organic Chemistry, Department of Biochemistry and Organic Chemistry, BMC, Box 599, Uppsala University, SE-751 24 Uppsala, Sweden. * Corresponding author:
[email protected]
ABSTRACT Heterodimerization between designed helix-loop-helix polypeptides was utilized in order to assemble gold nanoparticles on planar substrates. The peptides were designed to fold into four-helix bundles upon dimerization. A Cys-residue in the loop region was used to immobilize one of the complementary peptides on a maleimide containing SAM on planar gold substrates whereas the second peptide was immobilized directly on gold nanoparticles. Introducing the peptide decorated particles over a peptide functionalized surface resulted in particle assembly. Further, citrate stabilized particles were assembled on amino-silane modified glass and silicon substrates. By subsequently introducing peptides and gold nanoparticles, particle-peptide hybrid multi layers could be formed. Keywords: Heterodimerization, polypeptides, gold nanoparticles, four-helix bundle, helix-loop-helix, self-assembly
INTRODUCTION Self-assembly has emerged as one of the most promising and interesting strategies for nano-fabrication.[1-3] In contrast to conventional top down approaches, self-assembly is a spontaneous process where nano-sized building blocks goes from an unordered to a structurally more complex and ordered state under the influence of weak, non-covalent interactions. The building blocks can range from simple organic molecules to “large” inorganic nanoparticles, and by choosing building blocks with suitable properties, it is possible to control and dictate the processes of self-assembly in order to obtain complex architectures and functional nanostructures.[4-6] Biomolecular interactions are often highly specific and biomolecules therefore constitute a very attractive class of versatile building blocks in nanoscale self-assembling systems. Large biomolecules, such as antibodies, however often lack the robustness necessary required for applications in harsh environments, which may limit their use. For example, they may partly lose their three dimensional structure when adsorbed on surfaces and can in most cases not be repeatedly dehydrated without not losing activity. Designed polypeptides are therefore an interesting alternative, combining robustness with an enormous chemical and structural diversity. Designed polypeptides have been utilized in large number of organic self-assembling systems,[7-9] and for assembly of nanocomposite materials with many promising future applications.[10-12] Designed polypeptides with enzymatic activity and specific recognition and binding characteristics also have been reported on.[13, 14] We recently described how peptide dimerization and folding can be utilized for controlling the assembly of gold nanoparticles in
MEMS/MOEMS Components and Their Applications V. Special Focus Topics: Transducers at the Micro-Nano Interface, edited by Srinivas A. Tadigadapa, Babak A. Parviz, Albert K. Henning, Proc. of SPIE Vol. 6885, 688506, (2008) 0277-786X/08/$18 · doi: 10.1117/12.775806 Proc. of SPIE Vol. 6885 688506-1 2008 SPIE Digital Library -- Subscriber Archive Copy
suspension.[15] In this paper we report on how interactions between immobilized de novo designed polypeptides can be utilized to assemble gold nanoparticles on planar substrates. The optical properties of such gold nanoparticle assemblies are highly interesting for the development of Surface Enhanced Raman Scattering (SERS) active substrates and for providing substrates for Metal Enhanced Fluorescence (MEF).[16, 17]
RESULTS AND DISCUSSION In order to assemble dense layers of gold nanoparticles on planar substrates two designed heterodimerizing peptides were utilized. The two 42-residue helix-loop-helix forming polypeptides JR2EC and JR2KC were designed to heterodimerize at neutral pH.[18, 19] Dimerization induces folding from a random coil state into a molten globule-like four-helix bundle as schematically described in figure 1. Folding is driven by the formation of the hydrophobic core made up by the hydrophobic inner sides of the amphiphilic helices. At neutral pH, JR2EC and JR2KC have net charges of –5 and +11, respectively, and charge-charge interactions allow them to form heterodimers but prevent homodimerization. The dissociation constant (Kd) for dimerization in solution is ~ 0.02 mM.[19] Homodimerization of JR2EC, however, can be induced at pH 11.[20] Both peptide monomers have a Cys residue in position 22 located in the loop region, which enables site specific, thiol-dependent, immobilization onto a range of substrates. In addition to JR2EC and JR2KC, the two corresponding peptides JR2E and JR2K, where Cys was replaced by Val, were also synthesized. The amino acid sequences can be found elsewhere.[19]
f)
Figure 1. The helix-loop-helix polypeptides JR2EC (JR2E) and JR2KC (JR2K) are designed to heterodimerize at neutral pH. The peptides fold into a four-helix bundle upon dimerization.
JR2EC and JR2KC form sub-monolayers with an average thickness of 10-13 Å at neutral pH on bare gold (111) surfaces.[19] In the case of JR2EC, the ability to dimerize and fold is not significantly affected by immobilization whereas JR2KC adopt a conformation on bare gold that seems to hamper dimerization.[19] JRKC have a large number of Lys residues exposing primary amines which tend to orient towards and bind to the gold surface, thus obstructing the interaction with JR2EC. In order to reduce this effect, JR2KC was immobilized on a Self-Assembled Monolayer (SAM), containing maleimide-terminated EG3-disulphides. The double bond of the maleimide group undergoes an alkylation reaction with the thiol group in the peptides to form a stable thioether bond. To achieve a well ordered and protein repellent SAM with high binding capacity of the peptide, the maleimides were diluted with a hydroxylterminated EG3-disulphide. The largest amount of JR2KC could be immobilized at surfaces with a 1:4 ratio of maleimide- to hydroxyl-terminated disulphides, which resulted in an increase in thickness of 9 Å (Table 1). When treating the surfaces with JR2K, a significantly smaller binding of peptide was observed, indicating that the coupling mainly occurs through the thiol group of Cys. Using Surface Plasmon Resonance (SPR) (GE-Health Care, Biacore, Uppsala, Sweden) the ability of the immobilized peptide to bind the complementary peptide, JR2E, was confirmed (data not shown).
Proc. of SPIE Vol. 6885 688506-2
Table 1. The influence of surface density of maleimide groups on the amount of immobilized JR2KC
% of maleimide 0% 1% 10% 25% 100%
maleimides/nm2 0 0.05 0.5 1.25 5
peptides/nm2
∆d (Å) 0.6 2.5 5.0 9.2 6.7
0.01 0.05 0.09 0.17 0.12
fraction of occupied maleimide groups 100% 18% 14% 2%
The high net charge a JR2EC enables the formation of an extremely stable suspension when immobilized on gold nanoparticles. JR2EC was immobilized by incubating the as-prepared particles (~13 nm in diameter) in a 100 µM polypeptide solution over night. Excess peptide molecules were removed by repeated centrifugations reducing the concentration of unbound peptides to less than 0.5 nM. The particles were resuspended in 30 mM Bis-Tris pH 7 after each centrifugation step. Stable dispersions of particles functionalized with JR2KC could be prepared at high pH (pH 12) when JR2KC is homodimerized. At neutral pH the primary amines of JR2KC are protonated and the homodimer dissociates due to the destabilizing charge repulsion between the monomers, leading to a rearrangement of the monomers that resulted in loss of particle stabilization and flocculation. The dimerization between JR2EC and JR2KC was utilized to assemble gold nanoparticles onto planar substrates as schematically described in Figure 2A. A surface with JR2KC immobilized on a 25% maleimide SAM, was immersed in a dispersion of JR2EC coated particles (3 nM in 30 mM Bis-Tris pH 7) for 60 minutes, which resulted in a clearly visible extinction maximum in the UV-Vis spectrum (Figure 2B). The collective electron oscillations, or localized surface plasmon resonance (LSPR), in gold nanoparticles give rise to a strong extinction band in the visible spectra as described by Mie theory.[21] The resonance wavelength and bandwidth is dependent on the particle size and shape, the refractive index of the surrounding medium, and the temperature.[22, 23] When immobilized on a surface also the distance between the particles can have a major impact on the resonance conditions. In order to be able to use a conventional UV-Vis setup, very thin films of gold (10 nm) were evaporated on glass substrates. The LSPR peak position was found to be red shifted from approximately ~524 nm when dispersed in buffer to ~545 nm when immobilized on the surface. This shift is most probably induced by near field coupling between closely spaced particles in combination with the short distance to the gold substrate.
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