Enhanced Immobilization of Gold Nanoclusters on Graphite - The

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Enhanced Immobilization of Gold Nanoclusters on Graphite P. Rodríguez-Zamora, F. Yin, and R. E. Palmer* Nanoscale Physics Research Laboratory, School of Physics and Astronomy, University of Birmingham, Birmingham B15 2TT, United Kingdom ABSTRACT: The immobilization of individual biological molecules by metal nanoparticles requires that the particles themselves be immobilized. We introduce a new technique for immobilization of gold clusters based on their binding to small tunnels in a graphite support, themselves created by the implantation of small clusters. These tunnels are shown to perform as more effective cluster immobilization sites than point defects on the surface of graphite. The method is tested with atomic force microscopy (AFM) (both contact and noncontact mode) scanning. Size-selected clusters with 923, 561, 309, and 147 atoms have been immobilized and imaged with high-resolution, noncontact AFM.

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INTRODUCTION

sites for subsequent cluster deposition. The idea is to create cluster arrays more stable against AFM imaging. Experimental work and molecular dynamics (MD) simulations of the landing of size-selected clusters on the graphite basal plane at different impact energies have demonstrated that the clusters pin on the surface if deposition energy is higher than a certain energy threshold (Ec). The clusters implant onto the graphite and create amorphous carbon “nanotunnels” if the deposition energy is increased further above Ec. The depth and diameter of the nanotunnel can be manipulated by controlling the size of clusters and deposition energy.28−31 These nanotunnels then provide the binding sites for soft-landed, larger clusters. Apart from protein immobilization, this nanostructured platform may also have applications in electronics,32 catalysis,33 or plasmonics.34

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The immobilization of proteins by size-selected clusters is a relatively new route toward the study of single protein characteristics that has demonstrated advantages over homogeneous surfaces and other immobilization approaches, such as anchoring, orientation, and nondenaturation of single, isolated protein molecules.4−6 Single protein immobilization has been performed by means of various different methods to enable the study of individual protein properties,7 interactions,8 and the potential development of biological microarrays (biochips).9−13 One of the most versatile techniques for imaging individual proteins is atomic force microscopy (AFM), which has a broad range of biophysical applications, including single-molecule imaging,14−16 force spectroscopy,17,18 and real time protein− protein interactions19,20 and protein structural and biophysicochemical properties.21,22 An effective immobilization (tethering) technique is essential for AFM. Early experiments on protein immobilization with metal nanoclusters employed cluster self-pinning: the cluster deposition energy used fixed the clusters to their point of impact.23,24 However, the high kinetic energy also deforms the clusters, reducing their height after deposition. Soft-landed clusters, however, may undergo little shape deformation during deposition, but in the absence of a proper pinning site, tend to diffuse across the support25,26 and agglomerate at natural surface defects, compromising their utility for immobilizing proteins. As a solution to this problem, point defects created by argon ion irradiation were shown to provide a capable immobilization technique.27 Gold clusters were stable on graphite at least in noncontact AFM (NC-AFM) mode. However, the clusters trapped at argon defects were unstable against contact mode AFM (C-AFM) scanning. With the above background, we report in this article a new cluster immobilization approach, which involves the preimplantation of small gold clusters (20 atoms) to create binding © 2014 American Chemical Society

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EXPERIMENTAL SECTION The experimental methodology used in this work involved two steps: (1) the pretreatment of the graphite support by small Au cluster implantation and (2) the decoration of the treated graphite support by soft-landing clusters to create an array suitable for protein binding. All the size-selected Au nanoclusters and Ar ions were generated by a home-built RF (radio frequency) magnetron sputtering, gas condensation cluster source combined with a novel, lateral time-of-flight (TOF) mass filter,35 and deposited in high vacuum (∼10−7 mbar) onto precleaved highly oriented pyrolytic graphite (HOPG) samples. In step (1) of the method, Au20 clusters were implanted with an energy of 1.5 keV and density of 1.2 × 1010 clusters in a circular Special Issue: A. W. Castleman, Jr. Festschrift Received: January 26, 2014 Revised: May 4, 2014 Published: May 6, 2014 8182

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Figure 1. NC-AFM topographies of Au147 clusters soft-landed (500 eV) on a graphite surface decorated with point defects (twice the cluster density) created by argon ion sputtering at 500 eV. (a) Three-dimensional representation of two clusters; (b) broader survey scan; (c) histogram of the height distribution of the Au147 clusters; mean height 1.60 ± 0.42 nm.

area of diameter 4 mm (12.6 mm2), for every sample. In step (2), Aun clusters, with n = 147, 309, 561, and 923, were softlanded in the same deposition area with an energy of 0.5 keV in every case and with coverage approximately half that of the implanted Au20 clusters (6 × 109 in the same circular area). The sizes of the Aun clusters were selected so that their dimensions were smaller than those of single protein molecules. In order to compare the effectiveness of this new technique with the point defect (argon sputtering) technique, we also prepared different graphite samples by pretreatment with an Ar+ ion beam at 0.5 keV (1.2 × 1010 defects in 12.6 mm2) defects array. Again gold clusters with 147, 309, 561, and 923 atoms were subsequently deposited with an energy of 0.5 keV and with a coverage approximately half that of the Ar ions. The AFM images were acquired with a benchtop AFM (Park Systems model AFM-NSOM) at room temperature. In noncontact mode we used diamond-coated cantilevers with resonant frequency of 260−410 kHz (spring constant 21 N/ m), and in contact mode, silicon cantilevers with resonant frequency of 105−230 kHz (spring constant 1.75 N/m).

Table 1. Comparison between Experimental Cluster Heights Obtained by NC-AFM of Samples with Argon Defects Compared to Samples with Small Gold Cluster Tunnels; the Nominal Diameters of Spherical Gold Clusters Are Also Given cluster size

nominal diameter (nm)

Au147 AU309 Au561 Au923

1.7 2.3 2.6 3.1

experimental height (nm) Ar+ defects 1.60 2.09 1.95 2.36

± ± ± ±

0.42 0.30 0.31 0.66

experimental height (nm) Au20 channels 1.58 1.76 1.84 2.12

± ± ± ±

0.42 0.55 0.48 0.53

ambient conditions. Figure 1b shows a typical example of the random distribution of Aun clusters on a graphite surface preirradiated with argon ions. We did not observe accumulation of clusters at steps, which indicates that the Aun clusters were anchored on the defects created by Ar+ ion; the same was found with Au20 tunnels. The height of each size of Aun cluster soft-landed on both kinds of pretreated graphite samples was measured from the noncontact mode AFM images, and the corresponding mean values are listed in Table 1. These experimental heights were compared with the theoretical diameters of each size of cluster, calculated using the bulk (fcc) lattice spacing of gold, 4.08 Å (consequent nearest neighbor distance of 2.88 Å) and based on the dense packing of atoms in a spherical approach.36 For Aun deposited on both argon defects samples and Au20 tunnels

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RESULTS AND DISCUSSION AFM Characterization of Gold Clusters on Graphite. All Aun clusters soft-landed on both argon defects samples and Au20 tunnels samples were found to be stable against noncontact AFM scanning. This stability enabled the acquisition of NC-AFM images of size-selected gold clusters with size down to Au147 (Figure1a) at room temperature under 8183

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if the substrate−cluster interaction favors a truncated spherical geometry.39 The slightly higher distortion from spherical shape found for Aun clusters deposited on Au20 nanotunnel samples can be explained by the larger defect than that created by Ar+ sputtering (which removes only one carbon from the graphite lattice). The implantation of the small clusters creates nanotunnels on the graphite support with depth expected to be a linear function of the momentum of the Au20 clusters30 and with diameter approximately the cluster diameter. Such nanotunnels permit more C atoms to interact with the Au cluster atoms than point defects. Thus, it is not surprising that clusters soft-landed on the Au20 samples have a stronger interaction with the support40 and therefore decrease in height slightly compared with the clusters deposited on Ar+ defect samples. Testing Immobilization Efficiency of Small Clusters Tunnels versus Argon Defects. The immobilization of Aun clusters on a support was tested against the applied shear force that the AFM tip exerts on the clusters in contact mode.41 AFM contact scanning (with a constant applied force of 1 nN) of Au147, Au309, Au561, and Au923 clusters on Ar+ defects showed in every case that only one scan removed all clusters from the scanned region (Figure 2). Performing the same experiment on samples with Au20 nanotunnels revealed that many clusters remained in their original positions after C-AFM scanning (Figure 3). The behavior was found for each cluster size. However, as is also evident from Figure 3, the Aun clusters immobilized on the Au20 nanotunnels are not unperturbed by

Figure 2. NC-AFM topographies of soft-landed, size-selected Au561 nanoclusters deposited at 500 eV on graphite pretreated by Ar+ irradiation. The same sample (a) before and (b) after a C-AFM scan with applied force of 1 nN, performed in the marked subregion. The clusters inside the C-AFM area are dragged to the edge of the scanned zone.

samples, the experimental heights are close to but below the expected theoretical value for a spherical cluster. This discrepancy (4.2 and 6 Å on average for argon and Au20 samples, respectively, compared with the theoretical spherical value for the cluster diameters) can be explained by the limited but inevitable deformation that small metal clusters undergo on the surface after soft-landed even at very low deposition energies.37 The morphological changes of the clusters are likely due to a combination of plastic deformation induced by the graphite−gold impact38 and a partial wetting effect anticipated

Figure 3. NC-AFM topographies of soft-landed, size-selected Au923 nanoclusters deposited at 500 eV on graphite pretreated by implantation of Au20 clusters at 1.5 keV. (a) The same sample before and (b) after a C-AFM scan with an applied force of 1 nN, performed in the marked (solid-square) subregion in the same area. A number of clusters remain in their initial positions after the contact mode scan. (c) NC-AFM topography of the region marked by a dashed square in panel b, indicating that some dragging of clusters still occurs. (d) NC-AFM image inside the contact window shows that many clusters remain. 8184

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Figure 4. Effect of C-AFM on size-selected gold nanoclusters soft-landed on graphite predecorated with implanted Au20 clusters. (a,b) Height distributions of Au561 clusters before and after a C-AFM scan with applied force 1 nN. (c,d) Height distributions of Au923 clusters before and after a C-AFM scan with applied force of 1 nN. Mean cluster heights are reduced after contact mode and the standard deviations decrease. (e) Plot of the ratio of cluster heights before/after C-AFM scan for each size of cluster. (f) Plot of the ratio of cluster densities before/after contact mode scan ratio for each size of cluster. The increase in density is largest for the largest clusters.

result shows that the standard deviation of the height distribution of four kinds of immobilized Aun clusters reduced from ∼0.5 nm to ∼0.3 nm after one C-AFM scanning. This effect appears to represent a nanoplanarization44 action of the AFM tip upon the Aun clusters, a nanofabrication technique that is able to produce an array of clusters with the same height.

the contact mode scan. A decrease in average cluster height and an increase in density both occur, as evidenced by noncontact mode systematic characterization before and after a single contact mode AFM scan (normal force: 1 nN). The average height of the gold clusters Aun was reduced by a height ranging from 0.47 nm (for Au147 clusters) to 0.79 nm (for Au923 clusters); the height histograms for Au561 and Au923 are shown in Figure 4a−d as examples. The ratio of mean cluster heights, before and after contact mode, shows a monotonic relationship with the number of atoms in the cluster (Figure 4e). The change in cluster density before and after the contact scan shows a clearer dependency on cluster size (Figure 4f), being smallest for Au147 clusters, with 18% more clusters after C-AFM scan, 33% for Au309, 31% for Au561 ,and highest for Au923 with a 54% increase. The observed reductions in height suggest a measure of cluster dissociation or “shaving” under the AFM tip.42 The cluster fragments created by this action may then be trapped by vacant Au20 tunnels on the surface. This would explain the increase in density of clusters after the C-AFM scan. Further consequence of the contact mode scan of the pinned Aun clusters is the homogenization of the cluster heights. Given the probably nonspherical tetrahedral geometry of Au20,43 the orientation of the Au20 clusters when deposited determines the size of the damaged area, causing a difference in heights for the larger clusters immobilized by such defects. As shown in Figure 4a−d, we can see that the height distribution of immobilized clusters became narrow after C-AFM scanning. The statistical

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CONCLUSIONS We have developed a new methodology for the immobilization of (gold) clusters, with hundreds of atoms, on the graphite surface. The technique depends on the pretreatment of the graphite surface by the implantation of small gold clusters (20 atoms) to create tunnels, which provide binding sites for subsequently soft-landed Aun that show enhanced stability under AFM scanning in noncontact mode compared with the argon ion presputtering immobilization technique. Contact mode scanning of the immobilized clusters narrows the distribution of cluster height (nanoplanarization). This materials platform provides nanostructured surfaces with potential applications not only in protein immobilization, our initial interest, but also in nanoelectronics, nanophotonics, sensors, and catalysis.

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AUTHOR INFORMATION

Corresponding Author

*(R.E.P.) E-mail: [email protected]. Tel: +44 (0) 121 414 4653. Fax: +44 (0) 121 414 7327. 8185

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the EPSRC. P.R.-Z. thanks the Mexican National Council of Science and Technology (CONACyT) for Ph.D. studentship funding. R.E.P. is grateful for many valuable discussions over the past 20 years with Prof. Will Castleman and for his encouragement to pursue the cluster deposition problem.

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