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Aggregation Kinetics of Gold Nanoparticles at the Silica-Water Interface Ivo K. J. Kretzers, Robert J. Parker, Rouslan V. Olkhov, and Andrew M. Shaw* School of Biosciences, UniVersity of Exeter, Stocker Road, Exeter EX4 4QD, U.K. ReceiVed: October 21, 2008; ReVised Manuscript ReceiVed: December 12, 2008
The aggregation kinetics have been observed for citrate-reduced 15 nm gold nanoparticles at the native silica and modified silica-water interfaces. At the native, negatively charged silica-water interface, two-phase adsorption is observed: a pseudo-Langmuirian adsorption phase and, after an acid wash to remove the citrate ligand from the adsorbed particles, another pseudo-Langmuirian adsorption phase. A kinetic analysis of these phases shows an average adsorption rate constant of (1.5 ( 0.4) × 106 M-1 s-1 with no measurable desorption. Another acid wash induces a nearly linear aggregation phase with a 4-fold faster nanoparticles deposition rate. SEM imaging of the aggregation-phase surface shows the formation of small nanoparticle clusters of 10. The refractive index sensitivity of the produced surfaces was measured by varying the analyte refractive index at each phase: the aggregated clusters on the positively charged surface are up to 4 times more sensitive. 1. Introduction The design of biosensor surfaces based on nanoparticle architecture can exploit a number of surface enhancement effects associated with the target species being near the nanoparticle surface. The nanoparticle surface morphology and in particular the electric field strength of particular shapes, especially at the edges of the particles, have been employed in both surfaceenhanced Raman scattering (SERS)1,2 and surface-enhanced absorption spectroscopy (SEAS).3 The enhancement observed in both of these effects can be rather large with single-molecule detection reported for SERS enhancement at the surfaces between nanoparticles.4-7 An enhancement mechanism involving both an electric field and chemical effect have been invoked.2,4,8 We recently reported nonlinear protein adsorption kinetics9 for binding proteins to an 800-nanoparticle cluster showing sensitivity to attogram mL-1 protein concentration, which is a factor of 106 more sensitive than observed for a single particle. The electric field between the surface roughness features or aggregated nanoparticles is enhanced, coupling the radiation to the nanoparticle optical scattering10,11 properties allowing the nanometer-scale structure to act as a local optical aerial. These results begin to point toward an optimum particle size and structure or touching or nearly touching particles for biosensing.12,13 Nanoparticle syntheses of triangles, squares, rods, spheres, and octagons have been reported,10,14-16 but these show little improvement in the bulk RI sensitivity. Solution-synthesized nanoparticles are normally produced in the presence of ligand surfactant, which is responsible for maintaining the colloidal phase, and in the case of citrate-reduced colloids,17 the ligand provides a charged interface. While remaining on the surface of the particle the ligand might prevent interaction with binding proteins; therefore, it shall be removed when the particles are adsorbed to the sensor surface. The effects of field enhancement have been monitored using the total optical extinction of the nanoparticles with contributions from Rayleigh scattering and the localized plasmon excitation. * Corresponding author. E-mail:
[email protected].
The localized plasmon field penetrates approximately one particle radius into the medium above the particle and is sensitive to the local refractive index (RI). Biological events such as protein adsorption to the surface and protein-antibody binding have been observed by monitoring the change in the extinction of the metal nanoparticle using a number of techniques including evanescent wave cavity ring-down spectroscopy (e-CRDS).18-20 The nanoparticle surfaces used in e-CRDS experiments appear to be sensitive to changes of 10-4 RIU in the local refractive index although this depends on the wavelength of interrogation. This RI sensitivity may be compared with 10-6 RIU routinely achieved with continuous gold surface plasmon instruments.21-23 The adsorption of the nanoparticle to a sensor surface composed of native silica or modified silica requires the stabilized particle to bind preferentially to the surface. Binding kinetics have been observed previously for native silica surfaces24 for the citrate-reduced gold nanoparticles indicating a strong attachment to the negatively charged silica surface. The native silica surface has two Si-OH per nm2, which deprotonate as a function of the bulk pH, producing a negatively charged surface with a surface potential of -125 mV when fully dissociated.19,25 The charged surface attracts a bilayer of positively charged counter ions balancing the negative charge and producing a large concentration enhancement above in the interface.19 The charged bilayer around the nanoparticles may be reduced by the bulk ionic strength ultimately leading to the instability and the aggregation of the colloid. 24,26 The same destabilization appears to occur at the charged silica interface, resulting in a nanoparticle firmly adsorbed on the surface. The aggregation of nanoparticles in salt-destabilized solution was the mechanism used to generate the fractal clusters that demonstrated the nonlinear response to protein binding observed previously.9 However, controlling the aggregation process in solution is difficult because the kinetic process accelerates when nucleation around the seed particles occurs. Some interesting structures have been trapped during aggregation, including chains and cluster aggregates, but the interface offers a more
10.1021/jp809304z CCC: $40.75 2009 American Chemical Society Published on Web 03/17/2009
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controllable environment to moderate the growth of the aggregates. In this article, we present a series of aggregation experiments with citrate-reduced 15 nm gold nanoparticles adsorbing to the native silica and functionalized silica surfaces. The optical extinction of the surface has been followed in real time using e-CRDS, and the adsorption and aggregation kinetics have been observed directly. The silica surface may be readily functionalized with amine groups, producing a positively charged surface at which the negatively charged particles can aggregate. The aggregates have been imaged using SEM, and the resulting surfaces have been characterized for their refractive index and protein-binding sensitivity. 2. Experimental Methods The e-CRDS technique has been described in detail elsewhere11 and will be only briefly described here. Two highreflectivity (R ) 99.95%) mirrors are aligned opposite one another at a distance of 81 cm: one mirror is planar and the other mirror has a radius of curvature of 1 m, forming a stable optical cavity. Radiation from a diode laser (635 nm, bandwidth 5 nm) passes through a quarter-wave plate to control the plane of polarization and enters the cavity through the back of the planar mirror. A photomultiplier tube is mounted behind the concave mirror to collect the ring-down signal. The continuous wave diode laser is switched on and off at a frequency of 6 kHz, and the bandwidth is sufficient to overlap more than one cavity mode, allowing intensity within the cavity to build up to the maximum level determined by the cavity Q factor.19 The radiation intensity in the cavity decays when the laser is switched off with a characteristic ring-down time, τ, again determined by the cavity Q factor. A decrease in τ is observed if any radiation absorbing or scattering species are present in the cavity. A fused silica Dove prism is introduced into the cavity in order to study surface events. Both prism faces are antireflection coated to minimize the loss of light. A total internal reflection in the prism results in an evanescent wave propagating from the silica surface into the medium above with a penetration depth of ca. 200 nm fixed by the angle of incidence at the reflecting surface and the wavelength of radiation.19 Any species present within the evanescent field that scatter or absorb at the wavelength of the radiation will cause a decrease in the Q factor of the cavity and hence a decrease in τ. The prism is accompanied by a flow cell that allows the delivery of various solutions to the surface probed by e-CRDS with flow conditions that produce concentration-limited kinetics. The experiments were performed on two different prism surfaces: a negatively charged native silica surface and an insitu-prepared positively charged aminated surface. The quality of the surface is critical, and a stringent cleaning procedure was used: the Dove prism was incubated in aqua regia (1:3 v/v HNO3/38% HCl(aq)) for an hour, after which the prism was rinsed with an aqueous solution of Decon90, water, ethanol, and isopropyl alcohol (IPA) and finally wiped dry with lens tissue. The prism was then placed onto an optical mount in the middle of the cavity, and the flow cell (volume of 190 µL) was secured above the reflecting surface. The surface stability is checked by trailing τ while the prism surface is exposed to IPA and water. If required, an in situ amination of the prism surface was performed at this stage. After the ring-down time has stabilized, an aqueous solution containing sodium citrate at a concentration similar to that of the gold colloid (8.2 nM) is introduced into the flow cell prior to aggregation experiments. The aminated surface was prepared by allowing a 10% v/v solution of 3-aminopropylmethoxysilane in methanol to flow
Figure 1. Plot of λmax vs time for two citrate reductions of auric chloride: 5 mL of 90 mM sodium citrate (aq) added to 50 mL of 1 mM gold chloride (aq) at 94 °C; heating was stopped after 40 min. (b, O) Repeat experiments.
over the prism surface for 5 min. The surface modification was confirmed by changes in the crystal violet adsorption isotherm at neutral pH observed on bare and aminated silica surfaces.19 The native surface contains a number of silanol groups that become negatively charged when the bulk pH is >4. The addition of the positively charged crystal violet chromophore CV+ results in a large amount of optical absorbance, owing to the attraction of the positive ion to the negatively charged surface. However, the aminated silica surface is positively charged at neutral pH, and no CV+ absorbance was observed on the modified surfaces. The absorbance of 66 µM CV+ at pH 7 demonstrated a 2 orders of magnitude decrease when measured on bare and aminated surfaces, indicating the presence of a significant positive charge on the latter.19 Precise preparation of the gold colloid is critical to the reproducibility of the surface aggregation process. The colloid was prepared by the citrate reduction of HAuCl4.17 All glassware was washed prior to synthesis in aqua regia, then immersed in 0.1 M NaOH for 20 min, and subsequently washed in HPLC water and IPA and dried with nitrogen. Then, 50 mL of 1 mM HAuCl4 was degassed by bubbling nitrogen through the solution and heated to 94 °C while stirring. After 30 min, 5 mL of an aqueous solution of 90 mM sodium citrate was added, and the heating continued for another 40 min. UV/vis spectra were recorded at 5 min intervals for 50 min after the citrate addition, from which λmax was derived and plotted against time in Figure 1. The SEM images of the stable colloid show spherical nanoparticles with 15 ( 3 nm diameter.24 The colloid solution was diluted with 8 mM citrate to reduce the Au particle concentration to 0.35 nM before use in the aggregation experiments. The diluted solution was introduced into the flow cell, and the adsorption kinetics were monitored in real time as a change in the ring-down time of the cavity. Once the particles were adsorbed onto the surface, a washing cycle of sodium citrate, hydrochloric acid, and water was used to remove the citrate ligand from the surface of the nanoparticles. The refractive index sensitivity of the cleaned nanoparticle surface is obtained by measuring the change in extinction at 635 nm when the bulk solution in the flow cell is altered from water to IPA. An additional adsorption step is then possible by reintroducing the same colloid solution until the ring-down stabilizes. Washing and adsorption cycles were repeated until the extinction exceeded the dynamic range of the e-CRDS instrument, a total extinction of 10-2. The same cycles of washing and adsorption were performed for both the silica and
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Figure 2. Extinction at λmax ) 523 nm for three citrate-reduced gold colloid preparations showing two stable colloids (O and 0) and one intrinsically unstable colloid (b).
aminated surfaces, which were also removed from the cavity for SEM imaging at each stage of the surface aggregation.
Kretzers et al.
Figure 3. Adsorption kinetics of citrate-reduced gold nanoparticles (pH 7) onto the silica surface: (a) first-phase adsorption; (b) secondphase adsorption after the HCl wash at pH 2; and (c) third-phase adsorption after the second HCl wash showing an increase in the adsorption rate compared to that of the other phases. Arrows mark the colloid injection points. SEM images corresponding to each adsorption phase were taken in separate experiments. The inset shows a washing cycle sequence and an IPA/water switch to determine RIS and nRIS.
3. Results and Discussion The formation and stability of the colloid lead to aging processes in solution from natural aggregation and clustering. However, these experiments have concentrated on the formation of SERS or SEAS sites on the prism by aggregation processes at the surface measuring the sensitivity of the resulting biosensing surface by their refractive index sensitivity and protein binding. Colloid preparation is controlled by monitoring the position of λmax in the extinction spectrum as a function of time (Figure 1). The colloid changes slowly from the featureless spectrum of the auric chloride that develops the spectrum of the particle plasmon after about 10 min. The λmax of the broad peak decreases over a period 5 min until it reaches a plateau at around 535 nm. The plateau phase may last for 10-15 min and despite precise synthesis conditions is not well-defined. Two variations in the plateau length are shown in Figure 1 and are indicative of an aggregation process as part of the particle synthesis. The colloid formation mechanism has been studied in detail elsewhere,27 and electron micrographs have been taken of the intermediate plateau growth phase indicating that the colloid starts with a large cluster of pseudoparticles of 100 nm diameter that reaches a critical size and surface charge then disperses into the nucleation particles to form the remainder of the colloid. The colloidal solution is stable with λmax ) 523 nm after 35-40 min. However, these numbers are not precisely determined by the synthesis conditions. Once formed, the colloids are usually stable over a period of 10 days as determined by the extinction at λmax (Figure 2), and the colloids that were used during this stable phase in these experiments. Some batches of colloid are unstable, and the extinction at λmax degrades continuously so that after 25 days the colloid has become completely unstable; one in five batches shows this inherent instability. The colloidal solutions, diluted in citrate to 5%, were introduced into the flow cell, and the changes in τ were converted to the extinction using the conventional formula.18 Typical experimental kinetic data along with SEM images of the surface at different stages is presented in Figure 3. The extinction in the first adsorption phase can adequately be described in terms of Langmuirian adsorption kinetics (Figure 3a). Using the extinction coefficient derived previously for the 15 ( 3 nm nanoparticles24 of (6.4 ( 1.6) × 107 M-1 cm-1
corresponding to a scattering cross-section of 24.3 nm2, the surface coverage may be estimated to be 1.2%, consistent with the SEM image particle density. Most adsorbed nanoparticles at this stage are well separated from each other. The surface clearly reaches a steady-state population of charged nanoparticles during the first adsorption phase, resulting in a maximum extinction value of ca. 1.5 × 10-3. The surface is then washed in water, IPA, and HCl to remove the citrate ligand from particles, effectively removing the surface charge. There is little or no wash-off of the nanoparticles observed, and the removal of the citrate ligand should remove the charge from the surface. Clearly, subsequent interactions of colloid particles with the surface now include possible aggregation around the bare gold nanoparticles. A second adsorption phase is then possible with a similar adsorption profile reaching an extinction level of 3.8 × 10-3, which corresponds to ca. 3.0% surface coverage (Figure 3b). The SEM image of the surface at this point shows not only larger surface coverage but also an increased number of small aggregates, predominantly dimers and occasionally trimers. A second acid wash of the surface allows a third adsorption phase (Figure 3c), with a radically faster adsorption rate and significant cluster population on the surface as seen from the corresponding SEM image. The surface coverage estimated on the assumption that the extinction coefficient does not vary significantly for aggregates is 7.2%. The number of pseudo-Langmuiran steps varies between three and five before the onset of the nearly linear aggregation phase. Similar adsorption experiments were performed with colloids of differing age as monitored by the variation in λmax in the absorption spectrum. The refractive index sensitivity of the surface at each stage of the gold nanoparticles adsorption was determined according to the following expression
RIS ) √2σε
|
RI1 - RI2 ε1 - ε2
|
(1)
where ε is the extinction and σε is its standard deviation. Subscripts 1 and 2 correspond to analyte solutions with different
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TABLE 1: Values for RIS, nRIS, and ka Derived from Two Experiments of Multiphase Adsorption of Gold Nanoparticles on a Bare Silica Prism Surfacea ka/105 M-1s-1
linear slope/10-6 s-1
11.5 12.2 16.5
12 ( 2 18 ( 2
0.8 (0.1 1.4 ( 0.1 4.5( 0.1
14.9 12.4 11.8 11.1 18.1
14 ( 3 9(4 15 ( 5 6(3
0.4 ( 0.1 0.3 ( 0.1 0.3 ( 0.1 0.4 ( 0.1 6.1 (0.1
adsorption step
RIS/10-4
nRIS/RIU-1
Experiment 1 phase 1 phase 2 phase 3, (aggregation)
6.1 5.4 23
Experiment 2 phase 1 phase 2 phase 3 phase 4 phase 5, (aggregation)
14 8.4 7.4 5.9 18
a
Experiment 1 is illustrated in Figure 3. Errors are standard deviations of fits.
TABLE 2: RIS, nRIS, and Adsorption Gradients for the Gold Nanoparticle Colloid Adsorption to the Aminated Silica Surfacea colloid age/days
RIS/10-4
nRIS/RIU-1
linear slope/10-6 s-1
1 2 5
4.0 17 8.0
47.6 37.3 28.8
10.1 ( 0.6 12.4 ( 0.6 6.9 ( 0.5
a
Errors are standard deviations of linear fits.
bulk refractive indexes; water and IPA were often used in the present experiments, ∆RI ) 0.0445. This measure of the refractive index sensitivity may be compared for a number of surfaces and instruments: e-CRDS measurements24 of a nanoparticle fabricated surface give RIS ≈ 10-5 RIU at 635 nm and 10-4 RIU at 830 nm, compared with continuous gold surface SPR platform sensitivities of 70 RIU-1, but such values are not typical and practically are not possible to reproduce because of the highly uncontrollable character of the aggregation process. These results may be compared with those obtained from the previous study,9 where preaggregation in solution led to increased RIS and protein-binding sensitivity. 4. Conclusions Colloidal gold nanoparticles can be readily deposited on surfaces at the silica-water and modified silica-water interface with faster aggregation occurring at the positively charged interface. The refractive index sensitivity of the particle clusters is greater with an increased number of particles in the cluster, which may result from two factors: increased scattering crosssection and/or increased sensitivity associated with local field enhancement factors at the nanocluster surfaces. Acknowledgment. The work was supported by the RCUK, Basic Technology Grant EP/C52389X/1.
Figure 5. SEM image of the citrate-reduced gold nanoparticle adsorption to an aminated silica surface.
4. There is an immediate marked contrast to the adsorption observed on the bare silica, not with pseudo-Langmuirian adsorption but with single-phase nearly linear aggregation. Comparison of the initial slopes for the adsorption and aggregation phases (Tables 1 and 2) indicates that aggregation occurs approximately 4 times faster for the same colloid concentration. The adsorption rates on aminated surfaces are ca. 1.5-4 times faster than those observed in aggregation phases on bare silica surfaces, although the older colloids (greater that 5 days) show a slower adsorption rate than do the younger colloids (Table 2). Indeed, the positive surface charge must assist the attraction of negatively charged nanoparticles to the surface and also counteract the interparticle repulsion, which should first lead to a more packed adsorption monolayer and should then enhance the aggregation processes. Figure 5 shows an SEM image of the aminated silica surface taken after the gold nanoparticle adsorption experiment: the majority of the particles are deposited in large cluster aggregates containing tens of particles. The proposed mechanism of the aggregation on the bare silica surface is supported by the observation of the aminated surface aggregation kinetics. The positively charged aminated surface attracts the negatively charged nanoparticles, leading to rapid interfacial ionic strength-induced aggregation. This mechanism is also corroborated by the concentration dependence of the
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