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Polypeptide Folding-Mediated Tuning of the Optical and Structural Properties of Gold Nanoparticle Assemblies Daniel Aili,†,‡,|| Piotr Gryko,†,‡ Borja Sepulveda,^ John A. G. Dick,†,‡ Nigel Kirby,z Richard Heenan,# Lars Baltzer,3 Bo Liedberg,||,O Mary P. Ryan,†,* and Molly M. Stevens*,†,‡,§ Department of Materials, ‡Institute for Biomedical Engineering, §Department of Bioengineering, Imperial College London, Exhibition Road, SW7 2AZ London, United Kingdom Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Research Techno Plaza, Sixth Storey XFrontiers Block, 50 Nanyang Avenue, 637553 Singapore ^ Research Center on Nanoscience and Nanotechnology (CIN2) CSIC, Campus UAB Edificio Q third floor, 08193 Bellaterra, Barcelona, Spain z Australian Synchrotron, Clayton, Victoria 3168, Australia # ISIS, Rutherford Appleton Laboratory, Oxfordshire, United Kingdom 3 Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, 751 23 Uppsala, Sweden O Division of Molecular Physics, Department of Physics, Chemistry and Biology, Link€oping University, 581 83 Link€oping, Sweden
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bS Supporting Information ABSTRACT: Responsive hybrid nanomaterials with welldefined properties are of significant interest for the development of biosensors with additional applications in tissue engineering and drug delivery. Here, we present a detailed characterization using UVvis spectroscopy and small angle X-ray scattering of a hybrid material comprised of polypeptide-decorated gold nanoparticles with highly controllable assembly properties. The assembly is triggered by a folding-dependent bridging of the particles mediated by the heteroassociation of immobilized helixloophelix polypeptides and a complementary nonlinear polypeptide present in solution. The polypeptides are de novo designed to associate and fold into a heterotrimeric complex comprised of two disulfide-linked four-helix bundles. The particles form structured assemblies with a highly defined interparticle gap (4.8 ( 0.4 nm) that correlates to the size of the folded polypeptides. Transitions in particle aggregation dynamics, mass-fractal dimensions and ordering, as a function of particle size and the concentration of the bridging polypeptide, are observed; these have significant effects on the optical properties of the assemblies. The assembly and ordering of the particles are highly complex processes that are affected by a large number of variables including the number of polypeptides bridging the particles and the particle mobility within the aggregates. A fundamental understanding of these processes is of paramount interest for the development of novel hybrid nanomaterials with tunable structural and optical properties and for the optimization of nanoparticle-based colorimetric biodetection strategies. KEYWORDS: Gold nanoparticles, polypeptides, folding, helixloophelix, small angle X-ray scattering (SAXS)
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elf-assembly has emerged as a promising route for fabrication of nanomaterials with novel properties suitable for applications in tissue engineering,13 therapeutics,4 drug delivery,5 and biosensing.6,7 A key issue in the design of materials that are chemically and structurally defined at the nanoscale is the requirement of reproducible methods for precisely controlling the assembly process and the spatial arrangement of the components. A promising route to guide the assembly of nanosized inorganic building blocks is through the use of biological or biomimetic macromolecules such as DNA,8,9 proteins,10 and polypeptides.11,12 Biomolecules provide chemical and structural flexibility along with highly controllable and programmable intermolecular interactions. This has enabled the specific, controlled, and reversible assembly of a plethora of different r 2011 American Chemical Society
inorganic nanomaterials, including gold nanoparticles (Au NPs). The presence of order in nanoscale assemblies often relies on very homogeneous and monodisperse particles along with strong interaction potentials. However, soft interaction potentials typically found in biological and biomimetic systems, where the interaction energy is on the order of thermal energy, still allow for spontaneous order formation despite certain defects and polydispersity of the constituent elements.13 The utilization of biomolecules to guide the assembly of inorganic nanoscale building Received: October 10, 2011 Revised: October 31, 2011 Published: November 02, 2011 5564
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Nano Letters
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Figure 1. (a) Schematic illustration of the formation of the heterotrimeric complex formed as a result of the heteroassociation between the lysine-rich, nonlinear, 84 residue polypeptide JR2KC2 and two complementary glutamate-rich helixloophelix polypeptides (JR2EC). JR2KC2 and JR2EC are shown in blue and red, respectively. The peptides fold into two disulfide-linked four-helix bundles upon association. (b) Addition of JR2KC2 to Au NPs modified with JR2EC induces a rapid and folding dependent bridging aggregation of the particles.
blocks thus presents a powerful approach to control the assembly and introduce order at the nanoscale in hybrid materials. Au NPs are of particular interest in hybrid materials because of their optical properties that enable a convenient transduction mechanism for biosensor applications, based on the difference in color of dispersed and aggregated particles. DNA and polypeptide-modified Au NPs have consequently been widely utilized for colorimetric detection of a broad range of analytes,14 including metal ions,15 nucleotides,16 and proteins.1719 The distinct color of Au NPs is the result of collective electron oscillations, known as localized surface plasmon resonance (LSPR). The LSPR gives rise to a pronounced extinction band in the visible wavelength spectrum, and the position and width of the LSPR band are highly affected by particle aggregation. Particle aggregation results in a spectral red shift and broadening of the peak. The magnitude of this shift depends primarily on particle size, aggregate density, fractal dimensions, and aggregate size,20,21 whereas the rate of change of the spectral shift depends on aggregate size and growth rate. Information on the aggregate structure can be utilized to better understand and tune the optical properties of Au NP assemblies but can also be compared to common colloidal aggregate growth models,22,23 such as diffusion-limited colloid aggregation (DLCA), reaction-limited colloid aggregation (RLCA), and compact cluster (CC) formation, in order to better understand the interactions involved in the assembly process. We have previously described a strategy for controlled and reversible assembly of Au NPs based on a folding-dependent bridging using de novo designed polypeptides as schematically described in Figure 1.24 We have also recently employed this material in an assay for robust colorimetric detection of phospholipase activity.17 Here we present an in depth investigation of the optical and structural properties of this hybrid nanomaterial using small angle X-ray scattering (SAXS) combined with UVvis spectroscopy that provides a better understanding of the assembly process and information on how to tune and control these properties by employing highly specific biomolecular interactions. The polypeptide that was immobilized on the Au NPs is a glutamic acid-rich 42 amino acid helix loop-helix polypeptide (JR2EC). JR2EC have no ordered secondary structure at neutral pH but homodimerize and fold into a four-helix bundle at slightly acidic pH or in the presence of a range of divalent cations.25,26 A cysteine residue was included in the loop region to facilitate directed immobilization on Au NPs. In solution, two JR2EC monomers can heteroassociate with a second polypeptide
(JR2KC2) and fold into two disulfide-linked four-helix bundles as schematically illustrated in Figure 1a.24 JR2KC2 is a nonlinear 84 amino acid polypeptide rich in lysine residues, designed as two separate helixloophelix polypeptides (JR2KC) that are covalently linked by a disulfide bridge between cysteine residues located in the loop. Au NPs functionalized with JR2EC aggregate extensively and rapidly in the presence of JR2KC2.24 The assembly is mainly driven by the heteroassociation and folding of the polypeptides (Figure 1b). The influence of folding on the assembly of the particles was determined using a polypeptide with the identical amino acid sequence to JR2EC but all L-Ala were replaced by D-Ala, which effectively prevented this peptide from folding. This system thus offers an interesting and useful possibility to assemble nanoparticles by employing highly specific molecular interactions. In this work, we confirm that the particles form regularly ordered aggregates with well-defined particle spacing corresponding to the size of the folded four-helix bundles. There is also an intricate relationship between the dynamics, density and internal structure of the aggregates and hence their optical properties with the size of the Au NPs and the concentration of JR2KC2. These observations enable strategies for tuning of the morphology and the optical properties of gold nanoparticle assemblies, which can have a profound impact on their performance in colorimetric sensors and on the design of hybrid materials with a controllable spatial ordering at the nanoscale. This work is also of large interest for the fundamental understanding of the assembly processes of peptide-based nanomaterials and highlights a number of aspects that have a large influence on the dynamics and organization of nanoparticle assemblies. Results and Discussion. The 42-amino acid helixloop helix polypeptide JR2EC was immobilized on Au NPs with diameters of 10, 20, and 40 nm via a thiol residue in the loop region. The actual sizes of the as-received Au NPs were found to be 9.4 ( 0.8, 18.3 ( 2, and 37.8 ( 4 nm using SAXS, which correlated closely to the sizes obtained by transmission electron microscopy (TEM), 9.3 ( 0.7, 18.9 ( 1, and 38.4 ( 3 nm. The JR2EC polypeptides were immobilized under conditions yielding a relatively low surface coverage (∼60% of a monolayer) in order to facilitate the heteroassociation but also to minimize the influence of particle curvature on the surface concentration.27 The heteroassociation of JR2KC2 to the immobilized polypeptides was confirmed and characterized on planar gold surfaces using surface plasmon resonance (SPR), Figure 2. SPR was utilized to obtain the dissociation constant (KD = 0.3 μM) for the heteroassociation of JR2KC2 to JR2EC immobilized on gold. 5565
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Figure 2. (a) SPR response for the heteroassociation of JR2KC2 to JR2EC immobilized on gold surfaces, (black) 10 μM, (red) 1 μM, (orange) 0.5 μM, (yellow) 0.2 μM, (green) 0.1 μM, (purple) 50 nM. (b) Equilibrium response versus concentration. The solid line represents the theoretical fit for the equilibrium response as function of JR2KC2 concentration with the dissociation constant Kd = 0.3 μM, based on a bimolecular dissociation model.
This KD value is interestingly significantly lower than for the dimerization of the corresponding monomers in solution (KD = 0.02 mM),27 which could be due to the strong electrostatic interactions between the highly negatively charged surface and the positively charged JR2KC2 resulting in a pronounced preconcentration effect. When investigating the heteroassociation-mediated bridging of the gold nanoparticles, the absolute concentrations of the polypeptides were assumed to be more critical than the concentrations of nanoparticles. In order to keep the concentration of JR2EC constant in all samples and for all particle sizes, the concentrations of particles were thus adjusted with respect to surface area. In all measurements, the concentrations of particles were 1.6, 0.4, and 0.09 nM for 10, 20, and 40 nm Au NPs respectively, corresponding to a particle surface area per volume of suspension of 0.3 m2 L1, giving a concentration of immobilized JR2EC of approximately 70 nM. The concentration of unbound peptides was reduced to less than 0.1 nM by repeated centrifugations and exchange of buffer. The position of the LSPR band (λmax) of the dispersed particles was similar for all three sizes of particles in the size range used here (1040 nm), showing maxima at 523528 nm after peptide functionalization. Particle aggregation, induced by addition of JR2KC2, resulted in immediate and pronounced spectral changes including a red shift and broadening of the LSPR band with the optical shift varying significantly with particle size for the same concentration of JR2KC2. Representative UVvis spectra, before and after addition of 0.1 μM of JR2KC2, are shown in Figure 3. The 10 nm particles displayed homogenously red-shifted spectra, approximately 35 nm after about 20 min, and
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Figure 3. UVvis spectra of the JR2EC decorated Au NPs with radii of (a) 10 (b) 20, and (c) 40 nm before and after addition of 0.1 μM of JR2KC2. Spectra were recorded with 2 min intervals for 30 min. JR2KC2 was added after 6 min which resulted in a significant red shift and broadening of the LSPR band. Spectra before addition of JR2KC2 are shown in red and the last recorded spectrum for each particle size is shown in blue.
a slight broadening of the LSPR band accompanied by an increase in extinction. The changes in the spectra of the 20 nm particles were more pronounced, demonstrating a larger red shift (∼50 nm) and a significant broadening. The spectra of the 40 nm particles were further red shifted (∼120 nm) while having a broad shoulder remaining at the original LSPR peak position. The shift in the position of the LSPR extinction maximum (Δλmax) gives a good description of the optical spectral changes during the aggregation process but does not reveal any information about peak broadening. The changes in the ratio of the integrals from 700 to 550 nm and 540 to 490 nm, the so-called ΔA/D ratio, can be used as a highly sensitive and accurate method to also describe these spectral changes (Supporting Information Figure S1).28 ΔA/D and Δλmax as a function of JR2KC2 concentration and time for the different sizes of Au NPs are presented in Figure 4. The corresponding full UVvis spectra are included in the Supporting Information (Figures S2S4). The measurements were conducted before and directly after introducing JR2KC2 into the samples and spectra were subsequently recorded every two minutes for 30 min. The onset of aggregation was very rapid (
