Optical Sensitivity Comparison of Multiblock Gold–Silver Nanorods

Feb 21, 2013 - In this work, we tested multiblock nanorods (NRs) with Au and Ag segments for the surface plasmonic detection of dopamine (DA). A chang...
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Optical Sensitivity Comparison of Multiblock Gold−Silver Nanorods Toward Biomolecule Detection: Quadrupole Surface Plasmonic Detection of Dopamine Yoonjung Choi,† Jin-Ha Choi,§ Lichun Liu,† Byung-Keun Oh,*,§ and Sungho Park*,†,‡ †

Department of Chemistry, Sungkyunkwan University, Suwon 440-746, South Korea Department of Energy Science, SKKU Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea § Department of Chemical & Biomolecular Engineering, Sogang University, #1 Shinsu-Dong, Mapo-Gu, Seoul 121-174, South Korea ‡

S Supporting Information *

ABSTRACT: In this work, we tested multiblock nanorods (NRs) with Au and Ag segments for the surface plasmonic detection of dopamine (DA). A change in the quadrupole surface plasmon mode was found to be sensitive to the Au/Ag block length and relative block ratio in a single NR. The surfaces of the NRs were decorated with monoclonal antibody (Mab) against DA. By comparing the results for pure Au NRs with those obtained for multiblock Au−Ag−Au NRs, we found that the magnitude of peak-shifting for the multiblock NRs was much larger than that for pure Au NRs. This result was attributed to the higher sensitivity of Ag to a change in the dielectric constant of the surrounding medium when compared to Au and the sensitive surface plasmon coupling at the junction between Au and Ag blocks. The magnitude of peak-shifting was tuned as a function of both the length of the Ag block and the number of repeating units of Au and Ag in the NRs. KEYWORDS: dopamine, monoclonal antibody against dopamine, Au nanorod, Ag nanorod, Au−Ag−Au nanorod, quadrupole mode, optical property

1. INTRODUCTION

surfaces of nanoshells with other molecules that can interact with the analytes. In recent years, many studies on biomolecule detection have been conducted using anisotropic NPs, especially noble metal nanorods (NRs), as 1-dimensional (1-D) structures. This is because the dipole longitudinal mode, which results from the oscillation of electrons on the surfaces of NRs along the longer axis by an electromagnetic field, has much higher optical sensitivity than dipole transverse modes to a change in the refractive index of the surrounding medium.19−29 Due to the tunable sensitivity of the dipole longitudinal mode to a change in the surrounding medium according to the aspect ratio (A.R.) of the NRs, it has been considered that rod-shaped nanostructures are more suitable probes for sensing biomolecules by observing the change in their peak-position and intensity.19 NRs with different aspect ratios could be synthesized under the solution-phases and applied to detect and separate biomolecules in the solution. For example, Irudayaraj et al.20 and Wang et al.22 studied the detection of biomolecules using as-synthesized Au NRs that are stabilized by CTAB (cetyl trimethylammonium bromide) as a surfactant and

Electromagnetic waves can drive localized surface plasmon resonance (LSPR) at the surface of many types of metallic nanoparticles (NPs).1 LSPR has been found to be sensitive to the size, shape, and composition of NPs as well as to the refractive index of the surrounding medium.2 The chemical affinity of molecules to the surface of NPs can lead to a shift in the position of LSPR spectral bands that can be observed via UV−vis-NIR spectroscopy. Such a shift is induced by a variation in the refractive index around the NPs.3 This shift is directly proportional to the concentration of molecules within an effective detection range.4 Consequently, this label-free LSPR modality is useful in sensing applications and has spurred extensive research into a wide range of probe nanoarchitectures and target analytes.5−9 In previous research, spherical metallic NPs with isotropic shapes (0-dimension (0-D)) have been synthesized and employed as probes in LSPR sensing.10−16 Fujiwara et al.12 and Nath et al.11 fabricated spherical Au NPs and used them to detect biotin-streptavidin interactions, bovine serum albumin (BSA), and human serum albumin (HSA) by observing their LSPR bands. In studies conducted by the J. L. West17 and Sulabha K. Kulkarni groups,18 they detected immunoglobin and Escherichia coli using Au and Ag nanoshells after modifying the © 2013 American Chemical Society

Received: December 17, 2012 Revised: February 14, 2013 Published: February 21, 2013 919

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2. EXPERIMENTAL SECTION

stabilizing agent; a change in the LSPR band on the Au NRs was observed. However, NRs synthesized using CTAB surfactant cannot be directly applied in vivo and are prone to aggregate when the CTAB surfactant is removed from the surfaces of the NRs. Dopamine (DA) is one of the most important neurotransmitters in the nervous system, and an abnormal DA concentration in the brain may cause serious ailments such as Parkinson’s disease.13 Thus, it is important to detect the exact concentration of DA via a simple yet sensitive process. Although there are several innovative methods to detect DA using carbon-based nanoparticles,30 due to the electroactive nature of DA, electrochemical methods for DA detection have previously been established.31−33 However, a drawback in DA detection is that species coexisting with DA, such as ascorbic acid1,34 and uric acid (UA), interfere with the electrochemical signal.35 Since AA and UA are oxidized at potentials close to that of DA, an overlapping voltammetric response can lead to the false detection of DA. DA detection by colorimetric and spectrometric methods has recently been developed using metal NPs.13−15,36,37 In research by the Xiaogang Qu group, citrate-capped Ag nanospheres (NSs) were found to interact with DA via Ag-catechol interaction and agglomeration.37 Therefore, by adjusting the concentration of DA, the extent of Ag NS agglomeration could be controlled and a color change from yellow to brown allowed for the detection of DA. In work by the Shaogjun Dong group, Au NSs were synthesized from Au seeds using DA as a reducing agent.36 By adjusting the concentration of DA, the size of the Au NSs could be controlled and a change in the LSPR modes was observed via UV−vis spectroscopy. Most experiments on DA detection have focused on using spherical-shaped nanocrystals (NCs) and monitoring the change in peak-position and intensity with respect to the amount of DA molecules. In our previous work, the electrochemical deposition method was employed to fabricate various single-component NRs38,39 and multisegment NRs consisting of more than two different materials.40,41 The optical properties of the structures were studied in detail using a UV−vis-NIR spectrometer. In research by the Michael Natan and Christy Keating groups, multisegment nanowires with more than two different metal components were applied to detect oligonucleotides with various sequences on the basis on the chemical properties of each metal segment.43−49 The detection processes were conducted by observing the change in the refractivity of each metal segment or the decay of fluorescent molecules adsorbed on the surface of nanowires. Herein, we demonstrate that the quadrupole longitudinal mode from multiblock Au−Ag NRs is more sensitive with regard to biomolecular sensing than the quadrupole longitudinal mode from pure Au or Ag NRs. We fabricated Au−Ag−Au triblock and Au−Ag multiblock NRs by electrochemical deposition methods. The resulting NRs showed longitudinal quadrupole surface plasmon modes at a fixed aspect ratio. The surface of the NRs was modified with monoclonal antibody (Mab) against DA, and the variation in the magnitude of the quadrupole mode peak position was monitored as a function of the Au−Ag block ratio and block number as DA binding progressed around the NRs. As a control experiment, the results obtained with as-synthesized NRs were compared with those from single-component Au and Ag NRs with a similar aspect ratio.

Instrumentation. Field emission scanning electron microscopy (FESEM) images were obtained with JEOL 7000F and JEOL 7600F instruments. UV−vis-NIR absorption spectra were acquired using a Shimadzu UV-3600 spectrophotometer. Optical and fluorescence microscopy images were obtained with a Zeiss Axio Observer and Eclipse Ti-u (Nicon). AAO Synthesis. The synthesis procedure to produce anodic aluminum oxide (AAO) is based on a method developed by Masuda et al. (H. Masuda and K. Fukuda, Science, 1995, 268, 1466). An alumina plate (3.5 × 5.0 cm2) served as the anode and two graphite cells served as the cathode in a two-electrode electrochemical cell. A thin plate of high-purity (99.999%) alumina (Goodfellow Cambridge Limited) was electropolished in a mixture of absolute ethanol (Fisher Scientific Korea Ltd.) and perchloric acid (Junsei Chemical Co., Ltd.) in a volumetric ratio of 7:3 at 20 V for 2 min. In the first anodization step, a mirror-reflective aluminum plate was anodized in 0.3 M oxalic acid (Sigma-Aldrich) at 40 V and 0 °C for 12 h. The alumina layer was then removed in a mixture of 1.8 wt % chromic acid (Sigma-Aldrich) and 6 wt % phosphoric acid (Duksan) at 60 °C for 12 h. A second anodization step was then performed with 0.3 M oxalic acid at 40 V and 0 °C for 24 h to produce a highly ordered porous AAO template. The aluminum plate was removed by immersion in a saturated HgCl2 (Tedia) aqueous solution for 6 h, and a subsequent immersion in a 8.6 wt % phosphoric acid solution for 30 min was employed to widen the pores of the AAO. The pore diameter of the as-synthesized AAO was approximately 80 nm. Synthesis of Au and Au−Ag−Au NRs. The synthesis of Au and Au−Ag−Au NRs in this work was based on a method employed in our previous research.41 A thin layer of silver (∼300 nm) was first thermally evaporated on one side of the AAO template. This layer served as both a supporting layer for the deposited NRs and as a working electrode in a three-electrode electrochemical cell after being placed in physical contact with aluminum foil in a Teflon cell. An Ag/ AgCl reference electrode and Pt wire counter electrode were used to establish a three-electrode configuration. An Ag plating solution (Technic ACR silver RTU solution from Technic Inc.) was deposited into the nanopores of the AAO template at −0.95 V. For Au NRs, an Au plating solution (Orotemp 24 RTU from Technic Inc.) was deposited at −0.95 V. The length of the Au NRs was controlled by monitoring the charge passing through the cell. For Au−Ag−Au NRs, Au and Ag plating solutions were successively deposited at −0.95 V. The length of each Au and Ag segment was controlled by monitoring the charge passing through the cell. The Ag component was selectively etched with concentrated nitric acid (Samchun), and the template was subsequently immersed in a 3 M sodium hydroxide (Samchun) solution to dissolve the AAO template and release the synthesized NRs. The as-synthesized NRs were rinsed with distilled water until a pH of 7 was reached. The NRs were then redispersed in D2O (Cambridge Isotope Laboratories, Inc.) for UV−vis-NIR spectrum characterization. The resulting NRs were drop-cast on a highly oriented pyrolytic graphite (HOPG) film for FESEM imaging. A schematic representation of the fabrication process is illustrated in Figure S1 in the Supporting Information. Synthesis of Ag NRs. The synthesis of the Ag NRs was based on a technique employed in our previous research.39 A thin layer of silver (∼300 nm) was first thermally evaporated on one side of the AAO template. An Ag/AgCl reference electrode and Pt wire counter electrode were used to establish a three-electrode configuration. A Ni plating solution (Hantech) was deposited into the nanopores of the AAO template at −0.95 V, and the tips of the Ni nanorods were slightly oxidized by applying +0.95 V for 3 s in a 10 mM KOH aqueous solution. Next, a Ag plating solution (Technic ACR silver RTU solution from Technic Inc.) was deposited into the empty interior of the AAO template at a constant potential of −0.95 V. The length of the Ag NRs was controlled by monitoring the charge passing through the cell during electrochemical deposition. The AAO templates were subsequently dissolved in an aqueous 3 M NaOH solution. The resulting NRs were rinsed with distilled water until a pH 920

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Scheme 1. Modification of Au Single-Component and Au−Ag−Au Triblock NRs with DA Antibodies and DA Binding Process on the Surfaces of Au and Ag Blocks

Figure 1. The optical (A, C) and fluorescence images (B, D) after adsorbing DA antibodies tagged with 10-FITCs on bulk Au (left) and Ag (right) substrates (A, B) and on Au−Ag−Au triblock NRs (d = ca. 250 nm, L = ca. 10 μm) (C, D). The upper inset in part C is a FESEM image of asfabricated Au−Ag−Au triblock NRs. fragments. After incubation for 90 min at 37 ◦C, the residual 2-MEA was removed through dialysis (molecular cutoff membrane) against a PBS−EDTA buffer (pH 7.4), i.e., PBS with 5 mM EDTA. Antibody Immobilization on the NRs. A 10-fold diluted antibody fragment solution was first prepared using distilled water. Next, 10 μL of the diluted antibody fragment solution and 500 μL of an aqueous PBS (phosphate buffered saline) solution from SigmaAldrich were added to an Eppendorf tube containing as-synthesized NRs. The solution was subsequently mixed at 4 °C for 2 h using a mixer (My Lab SLRM-2M). After mixing, 500 μL of 3% BSA (bovine serum albumin) from Sigma-Aldrich in an aqueous PBS solution was added to the solution so as to block the remaining Au or Ag surfaces that were not completely covered with antibodies. This step is important to rule out the possible contribution from the adsorption of

of ∼7 was reached, and then an ultrasonic wave (40 kHz) was applied to the entire solution so as to detach the Ag NRs from the Ni/Ag junction. A bar magnet was immersed in the solution in order to collect Ni debris. The as-synthesized NRs were rinsed with distilled water and redispersed in D2O for UV−vis-NIR spectrum characterization. The resulting NRs were drop-cast on a HOPG film for FESEM imaging. Antibody Fragmentation. Antibody fragments were prepared through chemical reduction with 2-MEA (2-mercaptoethylamine) as a reducing agent. The method employed is similar to that described by Karyakin et al.42 Here, 2-MEA was applied to the antibody solution so as to fragment the dopamine antibody molecules (Abcam). During the incubation, a 2-MEA reduced disulfide bond located in the middle of the antibody served to separate the thiol-functionalized antibody 921

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Figure 2. The FESEM images (A and B) show Au single-component NRs (d = ca. 80 nm) with the average lengths of 200 and 596 nm, respectively. The insets show the dimensions of each block, where the lengths are represented below. UV−vis-NIR spectra (C and D) were corresponding to Au NRs shown in parts A and B. The black lines are corresponding to the spectra on original Au NRs, the red lines to the spectra on Au NRs after modifying the surface of NRs with DA antibodies, and the blue lines to the spectra on Au NRs after adding DA molecules into the solution. Each wavelength of peak-position is represented above the spectra. The longitudinal mode and the quadrupole mode of Au NRs were red-shifted (C) from 1110 to 1120 nm and (D) from 1340 to 1356 nm, respectively, after two steps. other molecules (i.e., not DA) on the NR surface. The final solution was mixed overnight (O.N.) at 4 °C, rinsed with distilled water, and ultimately redispersed in D2O for UV−vis-NIR spectrum characterization. Antibody−Antigen Coupling. A 10 μL, 1 mg/mL (6.53 × 10−3 M) aqueous DA (Sigma-Aldrich) solution and 990 μL of an aqueous PBS solution were added to the solution containing as-synthesized NRs with antibodies adsorbed on their surfaces. The final solution was mixed at room temperature (R.T.) for 1 h using a mixer. The mixture was redispersed in D2O for UV−vis-NIR spectrum characterization. A schematic representation of this process is shown in Scheme 1. Optical and Fluorescence Microscopy Imaging. All optical and fluorescence microscopy images were obtained using a Zeiss Axio Observer and Eclipse Ti-u (Nicon). A D1m optical/fluorescence microscope equipped with an AxioCam MRc 5 digital camera (filter sets of 45 HQ Texas Red shift free, 10 FITC shift free, and 49 DAPI shift free were used for red, green, and blue emission, respectively) was employed in this work. After several washing steps for the NRantibody complex, a small amount of ethanol was added to the complexes in order to dry out the remaining buffer solution and spread the complexes on a glass slide. These complexes were observed in optical and green mode using a light source and a suitable filter set.

and the central dark domain is comprised of Ag blocks. After modifying the NRs with the DA antibodies, all NRs show a green color in the fluorescence image (Figure 1D), indicating the successful modification of Au−Ag−Au triblock NRs with the DA antibodies. The representative FESEM images in Figure 2A and B show Au NRs (diameter ∼80 nm) with average lengths of 200 and 596 nm, respectively. The surfaces of the Au NRs were modified with the thiol-functionalized DA antibodies produced from the reduction of disulfide bonds (using 2-MEA as a reducing agent) linking two heavy chains of antibodies. To prevent the adsorption of molecules other than antibodies on the surface of the Au NRs, BSA was added to the solution to block the remaining Au NR surfaces that had not been covered with antibodies. During the experiment, the nonspecific (electrostatic) binding of BSA had a negligible effect on the optical properties of the Au NRs. Next, DA molecules were added to the solution, leading to the capturing of DA on the surface of the Au NRs via antigen−antibody interactions. We systematically observed the change in the LSPR bands of the Au NRs after each step. The obtained UV−vis-NIR spectra were normalized with the intensity of the transverse modes of pure Au NRs. As displayed in panel C, the dipole longitudinal mode of pure Au NRs was observed at 1110 nm before surface modification. After surface modification with the DA antibodies, the Au NRs showed negligible peak shifts (red traces). When DA molecules were introduced into the Au NR solution, a peak shift from 1110 to 1120 nm occurred, as evident by a blue trace, indicating the binding of DA to the surface of the Au NRs. However, the magnitude of the peak shift before and after the DA binding event is very small, approximately 10 nm. In panel D, the obtained UV−vis-NIR spectra correspond to those of the Au NRs (L ∼ 596 nm) in panel B. Before modification, the pure Au NRs showed typical LSPR modes: a transverse mode at 550 nm and a quadruple longitudinal mode at 1340 nm (black traces). The small peak at 894 nm is a higher-order longitudinal mode. As in the case of short Au NRs, the peak position of the quadruple longitudinal mode was shifted slightly to longer wavelengths (red traces). However, after adding DA

3. RESULTS AND DISCUSSION To evaluate the assumption of whether the surfaces of both the Au and Ag blocks in a single NR were successfully modified with thiol-functionalized Mab against DA, we tested the same surface modification scheme on bulk Au and Ag substrates. Here, Ag was first thermally evaporated on an Au substrate, and the substrate was subsequently immersed in a solution containing the DA antibodies tagged with fluorophores (10 FITC). The optical image shown in Figure 1A clearly reveals the color contrast between Au and Ag domains. The corresponding fluorescence image (Figure 1B) shows a homogeneous distribution of green color over the entire substrate, indicating that the surfaces of both the Au and Ag were successfully modified with the DA antibodies. Next, the same modification procedure was tested with Au−Ag−Au triblock NRs. The corresponding SEM images of Au−Ag−Au triblock NRs (diameter ∼250 nm, length ∼10 μm) are shown in the inset of Figure 1C. The two bright ends are Au blocks, 922

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Figure 3. The FESEM images of Au−Ag−Au triblock NRs. Total average lengths of triblock NRs are (A) 566 nm, (B) 589 nm, and (C) 546 nm, respectively. The insets show the dimensions of each block, yellow block for Au, and gray block for Ag, where the lengths are represented below.

Figure 4. The UV−vis-NIR spectra (A−C) were corresponding to the triblock NRs shown in Figure 3(A−C). The black lines are corresponding to the spectra on original triblock NRs, the red lines to the spectra on triblock NRs after modifying the surface of NRs with DA antibodies, and the blue lines to the spectra on triblock NRs after adding DA molecules into the solution. The wavelength of each peak-position is represented above the spectra. The quadrupole modes of the samples were red-shifted (A) from 1160 to 1181 nm, (B) from 1222 to 1245 nm, and (C) from 1088 to 1124 nm, respectively, after two steps.

molecules to the solution, the peak shifted from 1340 to 1356 nm (blue traces). The magnitude of the peak shift before and after the DA binding event was 1.6 times larger than in the case of shorter Au NRs, which demonstrates the higher detection sensitivity of longer Au NRs when compared to shorter ones. For the transverse modes, the peak shifts were negligible for both the short and long Au NR cases. In our previous work, we fabricated Au−Ag−Au triblock NRs and observed their LSPR bands resulting from coupling between Au and Ag blocks. The characteristic LSPR band of the Au−Ag−Au triblock NRs is quite similar to that of singlecomponent Au NRs when the aspect ratios of the NRs are similar.41 As a control experiment, we synthesized Au−Ag−Au triblock NRs and observed the change in their LSPR bands under the same experimental conditions described in the previous paragraph so as to compare the findings with those obtained with Au NRs. We varied the length of the Ag domains in the Au−Ag−Au triblock NRs at a fixed aspect ratio. Representative FESEM images (Figure 3) show a clear boundary between the Au and Ag blocks. The two bright ends of the NRs are Au, while the dark central block is Ag. The total average lengths of the triblock NRs in Figure 3A−C are 566 nm, 589 nm, and 546 nm, respectively. Shown in the insets of Figure 3 are the dimensions of each block, a yellow block for Au and a gray block for Ag; the lengths of each segment are

displayed below the illustration. A narrow size distribution for both the total length and the block length was observed. We observed the change in the LSPR bands of triblock NRs after surface modification with DA antibody and DA molecules. The UV−vis-NIR spectra corresponding to the triblock NRs in Figure 3A−C are shown in Figure 4A−C, respectively. The quadrupole modes of the samples in Figure 4A−C were observed at 1160 nm, 1222 nm, and 1088 nm, respectively. The mismatch in the peak positions for the samples was due to the slight difference in the total length of the NRs. As in the case of pure Au NRs, the peak position of the longitudinal quadruple mode showed very little shift after modifying the NR surface with DA antibodies (red traces in Figure 4A, B, and C). When DA molecules were introduced, the quadrupole modes were gradually red-shifted from 1160 to 1181 nm (Figure 4A), from 1222 to 1245 nm (Figure 4B), and from 1088 to 1124 nm (Figure 4C). As the length of the Ag domain increased, the magnitude of the peak-shift of the quadrupole mode increased. To verify whether or not the triblock NRs were broken during the experiment, FESEM images were obtained (Figure S2 in the Supporting Information). It was found that the triblock NRs were not broken but rather retained their structure after DA detection. On the basis of the UV−vis-NIR spectra shown in Figure 2D and Figure 4, the magnitudes of the quadrupole mode peak923

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shifting (Δλ) gradually increased from 16 to 36 nm as the y/x ratio of the NRs, namely the length of the Ag domain, increased. It is well-known that Ag has much higher optical sensitivity to a change in the refractive index of the surrounding medium when compared to Au.50 Therefore, when the Ag domain in the NRs was longer (high y/x ratio), a higher plasmonic shift could be observed due to the higher optical sensitivity of Ag when compared to Au. Single-component Ag NRs with a total average length of 606 nm were also fabricated using a method outlined in our previous work.39 The same procedure described above was then conducted so as to confirm whether the Ag NRs have the highest optical sensitivity among the as-synthesized NRs to the DA binding process (Figure S3 in the Supporting Information). However, the quadrupole mode of the Ag NRs was blue-shifted from 1306 to 1267 nm after modifying the surface of the Ag NRs with DA antibodies. The quadrupole mode was then redshifted to 1300 nm after DA molecules were added to the solution. The reproducibility of DA detection was also poor. There are several possible reasons for the abnormal behavior of the Ag NRs: (1) the nature of the Ag surface changed as antibodies were adsorbed onto the surface of the NRs due the relative instability of Ag in the same experimental environment, and (2) the quality of the Ag NRs is poorer when compared to that of the pure Au NRs and Au−Ag−Au NRs. Although there are several reports on using Ag nanoparticles for biosensor applications, Au nanoparticles are commonly employed in such a capacity due to the better environmental stability of Au NPs when compared to Ag NPs. In our case, we believe that the poor stability of the Ag NRs caused different peak shifts when compared to those of other NRs. To investigate whether the number of Au and Ag blocks in the NRs has any effect on the optical sensitivity of the NRs to detect DA molecules, two different multiblock NRs with alternating stacks of three Au domains and two Ag domains were fabricated, and the same experiment outlined above was

shifting (Δλ) for four different NRs were plotted as a function of the ratio of the Ag block length (y) to the total NR length (x); the results are displayed in Figure 5. The definitions of x

Figure 5. A plot shows the magnitude of the quadrupole mode peakshifting (Δλ) as a function of the ratio between the total length of Ag domain (y) and the total length of NRs (x). The upper inset shows a schematic illustration explaining the definition of x and y. A sample (A) is corresponding to Au single-component NRs in Figure 2A, and (B−D) are Au−Ag−Au triblock NRs in Figure 3(A−C). The y/x ratios of NRs are (A) 0, (B) 0.201, (C) 0.331, and (D) 0.659, respectively. The lower inset shows the illustration of four NRs with their proportional physical dimension. A scale bar is 600 nm in (A− D).

and y are given in the form of a schematic illustration in the upper inset of Figure 5. The y/x ratio of the Au single NRs in Figure 2B and the Au−Ag−Au triblock NRs in Figure 3 were 0 (Figure 5A), 0.201 (Figure 5B), 0.331 (Figure 5C), and 0.659 (Figure 5D). We could confirm that the magnitude of peak-

Figure 6. The FESEM images (A and B) show multiblock NRs having three Au domains and two Ag domains alternatively. The total average lengths of the samples are (A) 580 nm and (B) 563 nm, respectively. The upper insets show the dimensions of each block, yellow block for Au and gray block for Ag, where the lengths are represented below. UV−vis-NIR spectra (C and D) are corresponding to the multiblock NRs shown in parts A and B, respectively. The black lines are corresponding to the spectra on original multiblock NRs, the red lines to the spectra on multiblock NRs after modifying the surface of NRs with DA antibodies, and the blue lines to the spectra on multiblock NRs after adding DA molecules into the solution. The wavelength of each peak-position is represented above the spectra. The quadrupole modes of the samples were red-shifted (C) from 1157 to 1204 nm and (D) from 1127 to 1161 nm, respectively, after two steps. 924

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Au triblock NRs (Figure 7B). It is noteworthy to mention that the total portion of Ag for both NRs is the same, and the length of the Ag in both NRs is about 200 nm. The peak-shifting (Δλ = 34 nm) of the multiblock Au−Ag NRs is 11 nm larger than that (Δλ = 23 nm) of the Au−Ag−Au triblock NRs. Such a finding indicates that the plasmonic shift of the NRs is dependent on both the number of repeating units of Au and Ag domains in the NRs as well as the Ag block portion. As shown in a previous publication,40 the multiblock Au−Ag NRs act like single-component Au NRs in terms of the longitudinal surface plasmon band, regardless of the Au and Ag block length, because the coherent oscillation of surface free-electrons on the NRs actively occurs along the long-axis, which is described by surface plasmon coupling. The surface plasmon coupling between Au and Ag blocks would be affected by the change in the chemical environment that is induced by the adsorption of antigens and antibodies. A certain portion of surface free electrons on the Au and Ag blocks will participate in molecular adsorption, and, thus, the coherent oscillation of surface freeelectrons will be disturbed by a change in the local dielectric constant. When more junctions exist, it is possible that surface plasmon coupling will be more affected by molecular adsorption. On the other hand, when compared with the NRs in Figure 7D and E, which have three and five domains while the total length of the Ag domain (y) of the triblock NRs is longer than that of the multiblock NRs (y = 360 and 268 nm in Figure 7D and E, respectively), the peak-shifting (Δλ = 47 nm) of the multiblock NRs in Figure 7E is 11 nm larger than that (Δλ = 36 nm) of the triblock NRs in Figure 7D. With the above results, we could confirm that the more important factor in terms of the optical sensitivity of the NRs toward a change in the chemical environment is not the total length of the Ag domain (y), but rather the number of junctions in the multiblock Au−Ag NRs.

conducted using the as-synthesized multiblock NRs. The representative FESEM images in Figure 6A and B show multiblock NRs with total average lengths of 580 and 563 nm, respectively. The dimensions of each block are displayed in the insets of Figure 6A and B; the lengths are indicated below the illustration. Two multiblock NRs with different y/x ratios of 0.462 (Figure 6A) and 0.376 (Figure 6B), where x is the total length of the multiblock NRs and y is the sum of the length of each Ag domain, were synthesized. We observed the change in the LSPR bands of the multiblock NRs after surface modification; the UV−vis-NIR spectra obtained for the samples are plotted in Figure 6C and D. The quadrupole modes of each sample appeared at 1157 and 1127 nm in Figure 6C and D (black traces), respectively. We could confirm that both quadrupole modes were gradually red-shifted from 1157 to 1204 nm (Figure 6C) and from 1127 to 1161 nm (Figure 6D). It is obvious that the multiblock NRs with a y/x ratio of 0.462 (Figure 6A) show a higher plasmonic shift than the NRs with a y/x ratio of 0.376 (Figure 6B). As previously noted, the magnitude of peak-shifting increased as the y/x ratio increased because of the high optical sensitivity of Ag domains to the refractive index of the surrounding medium. To compare the multiblock NRs (Figure 6) with both the single-component Au NRs (Figure 2) and triblock NRs (Figure 4) in terms of optical sensitivity to the DA binding process, plots were generated to show the magnitude of quadrupole mode peak-shifting (Δλ) for five different NRs (Figure 7).

4. CONCLUSIONS In this work, we fabricated and tested multiblock NRs with Au and Ag segments for the surface plasmonic detection of DA molecules by observing the change in their quadrupole surface plasmon mode. The surface of the NRs was modified with Mab against DA for the detection of DA, by reducing disulfide bonds to make thiol-functionalized DA antibodies. By comparing the results for pure single-component Au NRs with those obtained for Au−Ag−Au triblock NRs, we found that the magnitude of the quadrupole mode peak-shifting (Δλ) for triblock NRs was much higher than that for pure Au NRs because of the higher optical sensitivity of Ag to a change in the refractive index of the surrounding medium when compared to Au and the sensitive surface plasmon coupling at the junction between Au and Ag blocks. However, single-component Ag NRs showed a different tendency in terms of peak-shifting when compared to Au and Au−Ag−Au triblock NRs because of the instability of Ag in the experimental environment and the low quality of the Ag NRs. The magnitude of the peak-shifting increased according to an increase in the length of Ag domains (y) and the number of repeating units of Au and Ag in the NRs, which is an important factor in the optical sensitivity of the NRs. The multiblock NRs, which show higher optical sensitivity for the detection of DA molecules when compared to either Au or Ag single-component NRs, are a good candidate to resolve the problems of Ag instability and the relatively low optical sensitivity of Au to the refractive index of the surrounding medium.

Figure 7. A plot shows the magnitude of quadrupole mode peakshifting (Δλ) on five different NRs. (A) Au single-component NRs shown in Figure 2B, (B) Au−Ag−Au triblock NRs shown in Figure 3B, (C) multiblock Au−Ag NRs shown in Figure 6B, (D) Au−Ag−Au triblock NRs shown in Figure 3C, and (E) multiblock Au−Ag NRs shown in Figure 6A, respectively.

Shown in Figure 7A−E is the peak-shifting (Δλ) from pure Au NRs (image shown in Figure 2B), Au-dominant Au−Ag−Au triblock NRs (shown in Figure 3B), multiblock Au−Ag NRs (shown in Figure 6B), Ag-dominant Au−Ag−Au triblock NRs (shown in Figure 3C), and multiblock Au−Ag NRs (shown in Figure 6A), respectively. A schematic illustration of the five different NRs is also displayed in the figure. When the total length of the five different-component NRs was fixed at about 575 nm, the magnitude of peak-shifting (Δλ) showed a different tendency depending on the block length and the number of junctions between Au and Ag blocks. As clearly displayed in Figure 7, the multiblock Au−Ag NRs (Figure 7C) exhibited larger peak-shifting than the Au-dominant Au−Ag− 925

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (B.O.), [email protected] (S.P.). Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (2012R1A2A1A03670370) and by the Pioneer Research Center Program (2012-0009565).

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