Method for the Generation of Surface-Bound Nanoparticle Density

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Method for the Generation of Surface-Bound Nanoparticle Density Gradients Renee V. Goreham,* Robert D. Short, and Krasimir Vasilev* Mawson Institute, University of South Australia, Mawson Lakes 5095, Australia ABSTRACT: We report a novel, versatile method for generating number density gradients of individual gold (Au) and silver (Ag) nanoparticles by a two-step method. First, a chemical gradient of amine surface functional groups is deposited by plasma copolymerization. Second, a density gradient of nanoparticles is formed by the immersion of the chemical gradient in solutions of nanoparticles. Chemical characterization by X-ray photoelectron spectroscopy and morphological analysis by atomic force microscopy shows that nanoparticle density closely follows the change in nitrogen surface concentration across the gradients. We also demonstrate that it is possible to control the slope of the gradients by using nanoparticle solutions of different concentrations. Important for technological and research applications, this method can be used with nanoparticles of various sizes and different materials. In addition, the use of plasma deposition allows such gradients to be generated on any type of substrate.

’ INTRODUCTION Over the past decade, there has been a growing research effort directed toward the development of new materials that display gradients of surface properties such as chemistry,1 wettability,2 biomolecules,3,4 and nanoparticles.5 Such materials are attractive in a number of fields, including cell biology, diagnostics and sensors, catalysis, and electronics.5-9 Surface gradients have also been utilized as engines to drive physical processes; for example, the directional movement of water droplets “uphill” following a gradient of surface energy.2 Gradients are also important in the field of biology. Many biological processes, such as chemotaxis, immune response, and cancer metastasis are driven by gradients of signaling biomolecules or extracellular matrix properties, but to date, the details of these processes remain poorly understood.10-14 Engineered surface gradients provide research tools to mimic and study these physiological processes in vitro using conventional analytical techniques.9 In addition, surface gradients can be designed and tailored in a fashion such that a single sample can be used to obtain multiple data points, which would traditionally require a large number of samples to be made. Thus, errors caused by sample reproducibility are eliminated, in addition to other benefits accrued, such as an increase in the speed of analysis. Gradients of nanoparticles are a particular class of gradients that have been utilized to explore the effects of nanoscale roughness on biological processes. For example, in a study of mammalian cell attachment, it was demonstrated that nanoscale surface roughness needs to be carefully considered because the number of attaching cells decreased markedly with increased nanoparticle surface density.15,16 Alternatively, as reported by Arnold et al.,17 gradients of nanoparticles can be used to provide anchorage points for the immobilization of cell-adhesion ligands, r 2011 American Chemical Society

allowing positioning cues for cell signaling, polarization, and migration. The ability to deposit nanoparticles with controlled density is also of interest in other advanced applications, including optoelectronic devices, catalysis, sensors, and high-efficiency solar cells.5-8 Several methods for generating gradients of nanoparticle density have been reported. Bhat et al.18 have fabricated gradients of amine-functionalized silanes and subsequently adsorbed gold nanoparticles through electrostatic binding. The same group also used polymer brushes of poly(acrylamide) with gold nanoparticles attached to the amine functional groups and deposited them in a gradient manner.7 Huwiler et al.19 developed nanoparticle density gradients by dipping a substrate (positively charged) into a solution of silica nanoparticles (negatively charged). Gradients of nanoparticle density have also been fabricated by low-energy cluster beam deposition.20 However, the above methods are only suited to single sample fabrication and are dependent on the supporting substratum material (e.g., silanes on silica, thiols on gold). Therefore, a simple and reliable method for generating gradients of nanoparticle density that can be applied to various types of substrate materials and to different size and types of nanoparticles has particular attraction. The goal of this work was to develop such a method that can be applied to any type of substrate material and used with nanoparticles of different composition and size. To this purpose, we have utilized plasma copolymerization to generate surface chemical gradients of nitrogen (N) functional groups (including amines),21,22 which are then used to adsorb surfaceReceived: November 24, 2010 Revised: January 18, 2011 Published: February 08, 2011 3429

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The Journal of Physical Chemistry C functionalized gold (Au) or silver (Ag) nanoparticles in a controlled manner. The use of plasma polymerization is attractive because the technique can be used to deposit thin functional polymer films on practically any type of substrate. It has been recently demonstrated that after the first few nanometers of film growth, the deposition process becomes entirely substrateindependent.23 Independence of the underlying material is particularly important in terms of using the same technique (reproducibly) for fabricating gradients on different substrata and for different assay formats.

’ EXPERIMENTAL SECTION Materials. Allyamine (AA) (98%, Aldrich), octadiene (OD) (98%, Aldrich), poly(vinyl sulfonate) sodium salt (PVS) (Aldrich), silver nitrate (Aldrich), sodium borohydrate (Aldrich), hydrogen tetrachloroaurate (99.9985%, ProSciTech), trisodium citrate (99%, BHD Chemicals, Australia Pty. Ltd.), and 2-mercaptosuccinic acid (97%, Aldrich), were used as received. For solution preparation and glassware cleaning, high-purity water was used, produced by the sequential treatments of reverse osmosis, two stages of mixed-bed ion exchange, two stages of active carbon treatment, and a final filtering step through a 0.22 μm filter. The final conductivity was less than 0.5 μS/cm with a surface tension of 72.8 mN/m at 20 °C. Fabrication of Chemical Gradients by Plasma Copolymerization. Gradients of OD and AA were plasma-deposited onto 13 mm glass coverslips using the method and apparatus described previously.21,28 A schematic of the apparatus is provided in the supporting material. The shape of the gradient was controlled by the rate at which the OD/AA flow rate ratio was changed. The initial flow rate of octadiene was 10 sccm, which was linearly reduced to 0 sccm between the 6 and 12 mm positions on the gradient surface. The flow rate of allylamine was linearly increased from 0 sccm at the 6 mm position to 10 sccm at the 13 mm position. The plasma was sustained using a 13.56 MHz radio frequency generator. A plasma of 10 W (dial value) was used and remained constant during the whole process of deposition. Synthesis of Gold Nanoparticles (AuNPs). AuNPs were synthesized by citrate reduction of HAuCl4. Particles of ∼15 nm diameter were synthesized from 150 mL of a 0.01% boiling solution of HAuCl4, to which 5.75 mL of a 1% solution of sodium citrate was added under vigorous stirring.24 The solution was left to boil for 20 min and then allowed to cool to ambient temperature. The AuNPs were then surface-modified with 2-mercaprosuccinic acid as in Zhu et al.25 The particle diameters were confirmed via AFM images. Synthesis of Silver Nanoparticles (AgNPs). AgNPs were synthesized as in Vasilev et al.26 In a typical synthetic procedure, 0.002 M/L of AgNO3 was dissolved in 50 mL of Milli-Q water in an Erlenmeyer flask. A 0.25 mg/mL portion of PVS was added under vigorous stirring. The role of PVS is to serve as a capping and stabilizing agent. After five minutes, 0.002 M/L of NaBH4 was added dropwise. The color of the solution changed from colorless to dark yellow-brownish within a few seconds. The synthesis was conducted at room temperature, and the AgNPs colloidal solutions were sealed and stored in the dark. These nanoparticles are stable for several months. Nanoparticle Adsorption. Nanoparticle deposition was carried out by the immersion of surface chemical gradients in a solution of nanoparticles either as originally synthesized or after

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dilution of 4 and 8 times. A deposition time of 1 h was used in the case of 15 nm AuNPs, 2 h in the case of 40 nm AuNPs, and 24 h in the case of silver nanoparticles (AgNPs). The deposition step was followed by a thorough washing with Milli-Q water to remove all weakly bound particles. X-ray Photoelectron Spectroscopy. XPS analysis was used to determine the surface composition of the octadiene-allylamine (OD/AA) gradients and the gradients with deposited Au and Ag NPs. XPS spectra were recorded on a Specs SAGE XPS spectrometer using a Mg KR radiation source (hν = 1253.6 eV) operated at 10 kV and 20 mA. Elements present in a sample surface were identified from the survey spectrum recorded over the energy range 0-1000 eV at a pass energy of 100 eV and a resolution of 0.5 eV. The areas under selected photoelectron peaks in a widescan spectrum were used to calculate percentage atomic concentrations (excluding hydrogen). High-energy resolution (0.1 eV) spectra were then recorded for pertinent photoelectron peaks at a pass energy of 20 eV to identify the possible chemical binding environments for each element. All the binding energies (BEs) were referenced to the C1s neutral carbon peak at 285 eV, to compensate for the effect of surface charging. The XPS analysis area was circular with a diameter of 0.7 mm. The processing and curve-fitting of the high-energy resolution spectra were performed using CasaXPS software. Atomic Force Microscopy. An NT-MDT NTEGRA SPM atomic force microscope (AFM) was used in noncontact mode to provide topographical images. Silicon nitride noncontact tips coated with Au on the reflective side (NT-MDT, NSG03) were used and had resonance frequencies between 65 and 100 kHz. The amplitude of oscillation was 10 nm, and the scan rate for 4 μm  4 μm images was 0.5 Hz. The scanner used had a maximum range of 100 μm and was calibrated using 1.5 μm standard grids with a height of 22 nm.

’ RESULTS AND DISCUSSION A schematic of our experimental approach is shown in Figure 1. In step 1, a chemical gradient of amine functional

Figure 1. Schematic representation of the strategy for preparing gradients of nanoparticle density: (1) Generation of the surface gradient via plasma copolymerization, (2) substrate is immersed into a solution of 2-mercaptosuccinic acid-modified colloidal AuNP’s and (3) formation of a number density gradient of nanoparticles by electrostatic adsorption. 3430

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The Journal of Physical Chemistry C groups is fabricated by the plasma copolymerization of AA and OD through a mask containing 1 mm slots onto 13 mm substrates held on a moving sample holder, as previously reported.22,27,28 First, OD (a pure hydrocarbon) is introduced into the reactor chamber. Through the slots in the mask, deposition starts at the 0 mm position onto the substrates. The sample holder is moved at a constant rate under the mask in the x direction from the 0 mm position to the 12 mm position. At a predetermined position (6 mm), the flow rate of OD is gradually reduced until it has completely ceased by the 12 mm position. While reducing the flow rate of OD, AA is gradually introduced into the plasma chamber until the flow rate of AA reaches 10 sccm at the 12 mm position. The resultant nitrogen surface gradients have been demonstrated to contain a population of amine surface groups, which have been utilized in previous studies to passively bind heparin21 and covalently immobilize poly(ethylene glycol).3 After the generation of these surface chemical gradients, they are immersed in a solution of AuNP’s for a predetermined time. The AuNP’s are surface-modified with 2-mercaptosuccinic acid.25 This modification provides on the surface of the AuNP’s carboxylic acid functional groups which in water carry a net negative charge. Our hypothesis is that within the nitrogen gradient, there is a gradient of protonated-amine functional groups, and thus, the negatively charged AuNP’s will electrostatically adsorb to these amines. This adsorption process will

Figure 2. Atomic ratios calculated from the XPS characterization. N/C atomic ratio across a pure chemical gradient (circles) and Au/C atomic ratio (squares) after the adsorption of AuNP0 s. The error bars represent one standard deviation taken from the first data point.29,30

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result in a higher density of AuNPs at the nitrogen-rich end of the gradient and in a lower density at the nitrogen-poor end. Figure 2 shows the chemical composition across the plasma polymer gradient as measured by X-ray photoelectron spectroscopy (XPS). The N/C atomic ratio before the adsorption of AuNPs, plotted on the left-hand axis, shows that the amount of nitrogen increases across the surface of the chemical gradient. Generally, the presence of nitrogen in the XPS spectrum is not sufficient evidence for the availability of amine groups; however, our earlier work with chemical gradients prepared by the same method has demonstrated the presence of surface amine functional groups distributed in a gradient manner.21,22 In the same figure, the Au/C atomic ratio after adsorption of 15 nm diameter AuNP’s is plotted on the right-hand axis. The atomic concentration of Au increases across the surface and follows a trend similar to that of the nitrogen. This is consistent with our hypothesis that AuNP’s would adsorb in larger numbers on the nitrogen-rich part of the surfaces and create a gradient of decreasing particle numbers toward the OD part. AFM imaging in tapping mode was used to directly visualize the nanoparticles surface density and to verify the particle diameter. Figure 3 shows representative AFM images at five positions on the surface (i.e., at the 2, 4, 6, 8, and 10 mm positions) starting from the nitrogen-poor end (left-hand side image) and finishing with the amine-rich end (right-hand side image). In this example, the nanoparticle diameter is 15 nm. In agreement with the XPS data, the AuNP density increases toward the nitrogen-rich end of the chemical gradient. It is worth noting that the nanoparticles deposit as individual nanoparticles and do not form aggregates, which is essential for any further uses of these gradients. In terms of potential application, it is also important that the methodology is applicable to nanoparticles of different sizes. Using the same strategy, we have prepared gradients of larger particles of 40 nm diameter. AFM images at five positions across the gradient are shown in Figure 4. As in Figure 3, the density of the nanoparticles increases from the amine-poor to the aminerich end of the surface. Furthermore, we aimed to demonstrate that we can control the slope of the gradient. We used the concentration of AuNP solution as a means to accomplish this goal. We found that to reduce the number of adsorbed nanoparticles for a given immersion time by approximately half (as determined by AFM imaging), we needed to dilute the original solution of AuNP’s by 4 times. Figure 5 shows the number of 15 nm diameter gold nanoparticles per an area of 5  5 μm across three gradients as quantified from the AFM images. Nanoparticles were deposited for a fixed period of time (60 min) from the original solution and after serial dilutions of 4 and 8 times. Figure 5 clearly shows that

Figure 3. AFM micrographs at positions 2, 4, 6, 8, and 10 mm across the surface of the nanoparticle gradient. Nanoparticle size is 15 nm. Adsorption was carried out from the as-synthesized nanoparticles solution without dilution. 3431

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Figure 4. AFM micrographs at positions 2, 4, 6, 8, and 10 mm across the surface of the nanoparticle gradient. Nanoparticle size is 40 nm. Adsorption was carried out from the as-synthesized nanoparticles solution without dilution.

Figure 5. Number of 15 nm gold nanoparticles across the gradient from the original (as synthesized) colloidal gold solution (circles) and after serial dilution of 4 (squares) and 8 (triangles) times. The size of the nanopaticles was 15 nm.

the density of the nanoparticles at a given position on the gradient decreased with reduced nanoparticle concentration. Error bars were generated by counting the particles in at least three images for any given position on the surface. The rather small error bars demonstrate the uniformity of the gradients produced by this method. These results show that by simply diluting the original solution, one can produce nanoparticle number density gradients in a controlled fashion. This method for generation of number density gradients of nanoparticles can be easily extended to other types of nanoparticles. An example with AgNPs is shown in Figure 6. AgNPs capped with sulfonic acid groups26 were used in the same fashion as the AuNPs. XPS analysis shows that the concentration of N, S, and Ag increase from the 1 to the 12 mm positions across the gradient. The sulfonic acid groups passivating the AgNPs carry a negative charge in water and, as in the case of AuNPs, lead to a larger number of nanoparticles adsorbed to the amine-rich end of the surface of the gradient. The use of plasma deposition to provide a surface chemical gradient makes our approach applicable to practically any type of substrate. Furthermore, the method used for generation of amine-functionalized surfaces gradients can be extended to generation of other surface functionalities. For example, surface gradients of carboxylic acid functional groups have been reported

Figure 6. XPS characterization of polyvinyl sulfonate-stabilized silver nanoparticles adsorbed across an N-functionalized chemical gradient. Atomic concentrations are shown for N (circles), Ag (squares) and S (triangles).

previously.28 These gradients could potentially be used for the immobilization of positively charged nanoparticles.

’ CONCLUSIONS In summary, we have developed a novel method for the generation of gradients of nanoparticle density. The method comprises two steps: first, the generation of a chemical gradients of amine functional groups via plasma deposition (plasma copolymerization); and second, the subsequent electrostatic adsorption of surface-functionalized, negatively charged nanoparticles to amine-functionalized gradient surfaces that contain a population of primary amines that readily protonate. Chemical characterization by XPS shows that the percent of Ag or Au on the nanoparticle gradients can be directly linked to the underlying surface nitrogen percent (before nanoparticle deposition). AFM imaging shows an increase in the number density of adsorbed nanoparticles toward the nitrogen-rich end of the gradients and that across (all) gradient surfaces, nanoparticles adsorb individually without aggregation. We also demonstrate the ability to control the slope of the nanoparticle gradients via the concentration of nanoparticles in solution. We show that our approach can be used with nanoparticles of various sizes and materials and, because plasma polymerization is used this method, can also be applied to different substratum materials. 3432

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’ AUTHOR INFORMATION Corresponding Author

*(R.V.G.) E-mail: [email protected]. (K.V.) Address: Mawson Institute and School of Advanced Manufacturing, Mawson Lakes Campus, University of South Australia, Mawson Lakes, South Australia 5095. Phone: þ61 8 83025697. Fax: þ61 8 83025689. E-mail: [email protected].

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