Two-Dimensional Ag Nanoparticle Tetramer Array for Surface

Article pubs.acs.org/JPCC

Two-Dimensional Ag Nanoparticle Tetramer Array for SurfaceEnhanced Raman Scattering Measurements Jing Chen,† Yongji Gong,† Jian Shang,† Jianlong Li,† Yu Wang,*,‡ and Kai Wu*,†,§ †

BNLMS, SKLSCUSS, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China § SPURc, 1 CREATE Way, #15-01, CREATE Tower, Singapore 138602, Singapore ‡

S Supporting Information *

ABSTRACT: A two-dimensional Ag nanoparticle tetramer array which served as a hotspot matrix for surface-enhanced Raman scattering detection of rhodamine 6G (R6G) molecules down to a concentration as low as 10−15 M was successfully fabricated by electrochemical deposition on an anodized aluminum substrate. The high detection sensitivity was attributed to both the electromagetic enhancement at the dense Ag nanoparticle tetramer hotspot matrix and chemical enhancement on the corrugated substrate. A single molecule dynamic adsorption behavior was experimentally sensed by the abrupt changes of the charateristic peak intensity and line shape in the spectroscopy when the R6G concentration was lowered to 10−15 M. Time-evolved spectroscopies revealed the adsorption behavior of either the single molecule in the nanogaps of 2−5 nm or multiple molecules in the nanogaps of 5−9 nm between the Ag nanoparticles.

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INTRODUCTION Since the surface-enhanced Raman scattering (SERS) measurements of single molecules1,2 were reported, extensive effort has been made for the analysis of single-molecule events3−11 and the efficiency improvement of single-moelcule SERS detection.12,13 Recent studies14−16 suggested that the single-molecule sensitivity was attributed to the hotspots offering ultrahigh enhancement.17 To the purpose of practical applications, cost-effective preparation approaches such as nanoparticle assembly18−20 and template-assisted preparation21−27 have been employed to generate hotspot-containing nanostructures. However, the fabrication of the SERS substrates with a high density of uniform hotspots is still a challenge.17 Recently, randomly stacked large-scale arrays28 of gold nanoparticles fabricated by the bottom-up approach have demonstrated huge field enhancements due to the strong coupling among spatially proximate adjacent arrays. Meanwhile, a three-dimensional (3D) hotspot matrix29 in a close-packed assembly of Ag nanoparticles shows a high enhancement effect in the moist state, benefiting from the high density of hotspots and uniformity of interparticle distance, in comparison to that in the dry state. However, the as-reported 3D geometry requires a liquid environment to prevent unexpected aggregation of nanoparticles, which would limit its practical application. On the other hand, a recent study by low-temperature tip-enhanced Raman spectroscopy (LT-TERS) showed that the Lorentzian line shape and narrow line width of the peaks in the Raman spectra were characteristics of single-molecule behavior of the rhodamine 6G (R6G) molecule, and these features could be © 2014 American Chemical Society

used to explore the interaction between the adsorbate and substrate.17 Therefore, how to fabricate a substrate that possesses a hotspot matrix of regularly arranged nanoparticles with a strong interhotspot coupling becomes the key to detecting single molecules. In this paper, we present a simple method to fabricate a twodimensional (2D) and large-area ordered Ag nanoparticle tetramer (AgNPT) array by electrodeposition on a hard template substrate, i.e., the anodized aluminum. Such a 2D AgNPT array could achieve a SERS detection limit down to 10−15 M, i.e., the single-molecule level. Variations in the intensity and line shape of the spectral features at various molecular concentrations were analyzed to address the molecule adsorption behavior. Time-evolved SERS spectra of the R6G molecules were also collected to explore the singlemolecule adsorption behavior. The origin of this high sensitivity was attributed to the collective enhancement arising from the AgNPT array serving as a 2D “hotspot lattice” and the chemical enhancement caused by R6G chemisorption on the roughened Ag surface as well.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. Ethanol (99%), silver nitrate (CP), and R6G (AR) were purchased from Beijing Chemical Works. Aluminum foils (0.5 mm thick, 99.99%) were Received: May 26, 2014 Revised: August 31, 2014 Published: September 4, 2014 22702

dx.doi.org/10.1021/jp505160e | J. Phys. Chem. C 2014, 118, 22702−22710

The Journal of Physical Chemistry C

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purchased from General Research Institute for Nonferrous Metals. 2.2. Preparation of the 2D AgNPT Array. An Al foil (0.5 mm thick, 99.99%, Research Institute for Nonferrous Metals) was annealed at 500 °C for 2 h in an atmosphere of N2 (30 sccm) and H2 (50 sccm) to increase the crystallization domain size. After cleaning, the Al foil was electrochemically polished at 20 V for 3−5 min in a solution of 20 vol % perchloric acid in alcohol and electrochemically anodized at 40 V for 3 h in 0.3 M oxalic acid solution, leading to an anodized aluminum oxide (AAO) layer on the Al foil. The AAO layer was afterward removed by immersing the oxidized Al foil in an aqueous solution containing 5% phosphoric acid and 0.2 M dichromic acid (1:1 in volumetric ratio) at 60 °C for 3 h, resulting in a hexagonal pattern of Al nanopits on the Al foil surface. The patterned Al foil was used as a cathode in subsequent electrochemical deposition of Ag nanoparticles (AgNPs). The electrochemical deposition was performed with a threeelectrode system monitored by an electrochemical workstation (CH Instrument, CHI 660B). A Pt foil served as the counter electrode and a saturated calomel electrode as the reference electrode. A salt bridge connected the reference electrode (immerged in 0.1 M KNO3 solution) and the work one (in 0.01 M AgNO3 solution). 2.3. Structural Characterizations. The morphologies of the as-prepared samples were characterized by a cold fieldemission scanning electron microscope (FESEM, Hitachi S4800) operated at 10 kV. The structure lattices were analyzed by a powder X-ray diffractometer (XRD, Rigaku, Dmax 2500) and a transmission electron microscope (TEM, FEI, Tecnai F20). The XRD data were recorded with a Cu Kα radiation source (λ = 1.54056 Å) at a scanning rate of 8°/min for 2θ ranging from 10° to 80°. For TEM measurements, the AgNPs electrodeposited on the porous Al substrate were carefully scraped and transferred onto a carbon-coated copper microgrid. 2.4. SERS Measurements. A 100 μL R6G aqueous solution with a concentration ranging from 2.4 × 10−8 to 2.4 × 10−15 M was dropped onto a series of carefully cut slices of the AgNPs on the aluminum foils (0.5 × 0.5 cm2) for SERS measurements. The SERS spectra were collected at room temperature on a Raman imaging microscope system (LabRAM, Horiba, HR800, with a laser source of 514 nm in wavelength) and on a Research Laser Raman microscope (RM1000, Renishow, with a laser source of 633 nm in wavelength). For the sample of 2.4 × 10−8 M, the incident laser light was 0.5 mW in power, and the spectral acquisition time was 1 s. For the sample of 2.4 × 10−15 M, the laser power was 0.05 mW (514 nm) and 0.1 mW (633 nm), respectively. The spectral acquisition time was 10 s with accumulation cycles of 10. The laser spot in all measurements was 1 μm in diameter with a 50× objective lens.

Figure 1. Schematic diagram of the fabrication procedure for the AgNP array (a)−(d) and corresponding FESEM image of the AAO layer (e), the porous Al substrate after removal of the oxide layer (f), and electrodeposited AgNP array (g). The red dots highlight the eventually developed AgNPs sitting on top of the vertices of the substrate.

hexagonally packed bowl-like nanopits of about 94 nm in diameter (Figure 1f) whose edges were curved to form sharp Al needles (vertices) at the connection points of three neighboring bowl-like nanopits. Finally, AgNPs and nanoedges were prepared at the sharp Al vertices and curved edges by electrochemical deposition (Figures 1d and 1g) by carefully controlling the deposition duration. Otherwise, random AgNPs and an Ag overlayer were formed on the Al substrate. The morphology of a typical and large-area 2D AgNP array was shown by the SEM image in Figure 2A, where the densely packed 2D AgNPs could be clearly seen. The 2D Ag array contained AgNPs of 45 nm in diameter (Figure 2B) and 80 nm in height (Figure 2C) connected by Ag nanoedges sitting on the porous Al surface with an interpore spacing of 94 nm (Figure 1f) under the electrodeposition condition of −1.0 V for 60 s. Obviously, the arrangement of the electrodeposited AgNPs duplicated the periodic (Figure 1f) and even wrinkled (see Figure S1 in Supporting Information) structures of the porous Al substrate. The domain size of the orderly packed AgNPs could be as large as tens of micrometers. The gaps between neighboring AgNPs were typically 2−10 nm (Figure 2D). The prepared AgNPs were crystallized, as shown by the XRD measurement (Figure 2F) of the scratched AgNPs from the substrate (Figure 2E) and high-resolution TEM (HRTEM) detection (Figure 2G). In the HRTEM image, the interplanar distances of the (111) and (200) faces were correspondingly measured to be 0.235 and 0.204 nm, in good agreement with their counterparts in large Ag crystals. 3.2. Typical SERS Spectra at Low Concentration. A series of SERS spectra on a densely packed 2D AgNP array for detection of the R6G molecules in solution with a concentration as low as 2.4 × 10−15 M had been collected. A typical set of the Raman spectra recorded with an incident laser light of 633 and 514 nm in wavelength is shown in Figures 3A and 3B, respectively. A significant enhancement of the Raman vibrational bands of the R6G molecule could be noticed (trace a in Figure 3A and trace c in Figure 3B) on the orderly packed AgNP array (Figure 3C) in comparison to those (trace b in Figure 3A and trace d in Figure 3B) collected on an irregular Ag substrate (Figure 3D). The backgrounds (in red) of the

3. RESULTS 3.1. Morphology and Structure of the Ordered 2D AgNPT Array. A typical electrodeposition experiment for fabrication of an ordered and closely packed AgNPT array was carried out at an electrodeposition voltage of −1.0 V for 60 s. A schematic diagram for the whole fabrication process is given in Figure 1. Initially, the annealed Al foil (Figure 1a) was electrochemically anodized to form a porous AAO layer (Figure 1b) whose top-view SEM image is given in Figure 1e. Afterward, the alumina layer was removed by acid etching (Figure 1c) to expose the bare Al surface containing 22703



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Figure 2. FESEM images of the AgNP array on aluminum surface, deposited at −1.0 V for 60 s. (A) SEM image of a typical AgNP array of tens of square micrometers in area. (B) Zoom-in top-view FESEM image showing the particle size. (B) Zoom-in side-view FESEM image showing the AgNP height. (D) Top-view FESEM image showing the nanogaps in between the AgNPs. (E) TEM image of the AgNPs scraped and transferred onto a carbon-coated copper microgrid. (F) XRD of the scraped AgNPs. (G) HRTEM of an individual AgNP.

Figure 3. Typical SERS spectra of 2 × 10−15 M R6G on the AgNP substrates. (A) λex = 633 nm, Pacq = 0.1 mW, tacq = 10 s; (B) λex = 514 nm, Pacq = 0.05 mW, tacq = 10 s. Background (red) calculated according to ref 30. Traces a and c are collected on the ordered 2D AgNP array (C). Traces b and d are the results on the irregularly packed AgNP film (D).

where ISERS and IRS refer to intensities of the selected bands in the SERS and non-SERS spectra, respectively; NSurf and NVol are the number of molecules contributing to the SERS and non-SERS signal, respectively. To estimate the enhancement effect of our prepared 2D AgNP array sample with respect to the roughened Ag film, we redefine EF as

Raman spectra on the densely packed 2D AgNP array were subtracted by using the method developed by Galloway et al.30 The enhancement factor (EF) of a sample can be determined by EF =

ISERS/NSurf IRS/NVol 22704



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Table 1. Summary of the Main Features Experimentally Detected in the SERS Spectra and the Theoretically Calculated Vibrational Modes with Potential Energy Distribution (PED) Values for the Moieties Present in R6Ga theory17

experiment I

position

II

position

position

cm−1

cm−1

cm−1

methyl

xanthene

ethyl

phenyl

ester

1649 1608 1452 1415a

1654 1579 1438 -

1347a 1306a -

1291

-

1209

1145

1148 1126# 995 971

1658* 1607 1579* 1561* 1515* 1450 1441 1424* 1412 1380 1356* 1314 1298* 1271 1195* 1174 1144 1129* 1049 989 959 925 826 772* 669 632 616*

8.9 0 1.8 1.3 11.8 0.3 37.8 37.9 0 3.3 10.8 0.4 1 0.7 0.4 0 0.1 13.5 92.8 0 16.8 1 0.2 16.8 22.5 0.4 3.5

89.6 3.6 11.5 73 28.2 2.1 11.6 30.3 0 22.8 49.8 9.1 34 70 61.2 0 15.4 27.5 7.1 0 18.2 40.8 98.2 42.3 71.7 66.8 39.2

1.3 0 86.1 24.2 58.1 1.3 47.9 17.1 0 73.9 28.4 87.5 60.9 17.5 38.3 0 79.2 40.5 0.1 0 3 32 1.6 39.7 5.8 3.7 6

0 96.2 0.5 1.5 1.8 95.9 2.7 14.5 0.5 0 11.1 3 2.4 9.3 0.1 100 5.1 16.1 0 99.9 62 22.8 0 1.1 0.1 25.7 51.1

0.2 0.2 0 0 0.1 0.4 0 0.2 99.5 0 0 0.1 0.8 2.5 0 0 0.2 2.4 0 0 0 3.4 0 0.2 0 3.4 0.1

1069 936 624

827 672 -

PED value of vibrational modes (%)

a

Notes: (1) Methyl, xanthene, ethyl, phenyl, and ester correspondingly refer to the methyl groups attached to the xanthene ring, the xanthene ring, the ethylamine groups attached to the xanthene ring (ethyl), the phenyl ring, and the ester group attached to the phenyl ring. (2) Superscripts I and II refer to experimental results measured by the excitation laser of 633 and 514 nm in wavelength, respectively. (3) Superscript “a” refers to the peaks which are also observed by LT-TERS, and big peaks in the TD-DFT spectra were marked by ‘*’.17 (4) Superscript ‘#’ marks the peak observed in all Raman spectra collected with a 514 nm laser.

EF =

IT/NT IR /NR

Table 1 summarizes the Raman feature assignments by referring to theoretical calculations of free R6G.17 The main vibrational features can be assigned as below. The feature at about 230 cm−1 is the vibration of the whole molecule against the Ag substrate, i.e., the Ag−N stretch.31 In the Raman spectrum collected with a 633 nm laser (trace a in Figure 3A), some weak peaks17 were enhanced on the 2D AgNP array substrate, i.e., features sitting at 1453 (phenyl ring) and 1416 cm−1 (ester group attached to the phenyl ring). In addition, some prominent features are missing in comparison with the calculated Raman spectrum of free R6G. On the other hand, for the Raman spectrum collected with a 514 nm laser (trace c in Figure 3B), the features at 1653 (xanthene ring) and 1579 cm−1 (ethylamine groups attached to the xanthene ring) become more intense. The major feature at 1209 cm−1 is likely due to the red shift of the band at 1195 cm−1 (ethylamine groups attached to the xanthene ring and xanthene) in the calculated Raman spectrum of free R6G. The features at 1305 (trace a in Figure 3A) and 1290 cm−1 (trace c in Figure 3B) correspond to the 1298 cm−1 in calculated spectrum of free R6G with potential energy distribution (PED) on the xanthene ring and its attached ethylamine groups. Generally, the mostly enhanced features in the Raman spectra collected with a 633 nm laser fall into the region of 1300−1500 cm−1, while the counterparts

where IT and IR represent the intensities of the selected bands in the SERS spectra for the 2D AgNP tetramer array sample and the disordered AgNP sample, respectively; NT and NR are the number of molecules contributing to the signals on the 2D AgNP array and disordered AgNP samples, respectively. To calculate the EF for our prepare 2D AgNP array, we selected three intense Raman features, namely, 1653, 1581, and 1290 cm−1, for the 2D AgNP array sample (Figure 4SA, Supporting Information) which correspond to 1645, 1574, and 1307 cm−1 features for the disordered Ag sample (Figure 4SB, Supporting Information). NT and NR were obtained by dividing the total number of molecules in the R6G droplet by the projected surface area of the sample and then multiplying by the excitation laser spot area, yielding 10−2 and 1010, respectively. The calculated EF for the features at 1653, 1581, and 1290 cm−1 are correspondingly 7.5 × 1011, 4.3 × 1012, and 3.7 × 1012. The EFs for these three features were averaged to be about 2.9 × 1012. The average EF was actually underestimated because the disordered AgNP film on the Al substrate should possess certain surface enhancement as well. 22705



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Figure 4. Spectral shift (A) and Raman line shape (B) of the features (the 1000−1800 cm−1 spectral region) in the SERS for R6G on an ordered AgNP array as a function of the solution concentration. (a) 2 × 10−15 M: λex = 633 nm, Pacq = 0.1 mW, tacq = 10 s (pink), (b) R6G 2 × 10−15 M: λex = 514 nm, Pacq = 0.05 mW, tacq = 10 s (dark cyan), (c) the same as in (b) but at a different site (blue), (d) 2 × 10−12 M: λex = 514 nm, Pacq = 0.5 mW, tacq = 1 s (red), and (e) 2 × 10−8 M: λex = 514 nm, Pacq = 0.5 mW, tacq = 1 s (black). All spectra were fitted and background-subtracted. The wavenumber of each feature is marked in (A), and its fwhm is given in (B). Colored dots are the experimental data and black curves the fitted results. All spectra were normalized to their maximum intensity feature. The fwhm values in parentheses in (B) indicate the uncertain results caused by their background fitting and subtraction.

with a 514 nm laser fall in the regions of 1500−1700 cm−1 and 1000−1300 cm−1 (traces a and b in Figure 4A and Figure S4A, Supporting Information). It turns out that the phenyl and the xanthene rings (and their attached groups) are most strongly enhanced with a 633 and 514 nm laser, respectively. These two sets of Raman feature profiles are complementary to each other, and their combination gives the full Raman spectrum of the R6G molecule in the regime of 1000−1700 cm−1. The wavelength-sensitive enhancements have been reportedly attributed to the optimized condition at which the wavenumber, λmax, of the localized surface plasmon resonance (LSPR) equals the excitation wavelength (in absolute wavenumber) minus half the Stokes shift of the involved band.32,33 The features between 600 and 700 cm−1 are the in-plane bending of the C−C−C ring in the R6G molecule.32 The main feature at about 937 cm−1 relates to the wagging of the phenyl ring. The 1069 cm−1 feature is localized in the phenyl region or is attributed to the CH2 twisting motion of the ethyl ester part of phenyl.34 The 1209 cm−1 feature is assigned to the vibrations relating to both xanthene and ethyl groups according to the calculations.17 The 1347 cm−1 peak is due to the aromatic C−C vibration.31 The 1415 cm−1 feature stems from the C−O vibration of the ester group. The features around 1600 cm−1 come from the ring breadth of the phenyl group. The 1649 cm−1 peak is the CC stretch of the xanthene group.31 It is noticed that the features at 1415, 1347, 1306, and 1302 cm−1 have also been detected by LT-TERS.17 For the Raman spectra collected with a 633 nm laser, the features with PED on all groups such as 1579, 1561, 1515, 1424, 1129, and 772 cm−1 were absent. For the spectra collected with a 514 nm laser, lost were the features with PED on the whole R6G such as 1561, 1515, 1356, and 772 cm−l. To verify that the Raman spectra did come from the R6G molecule, we repeated the spectral measurements at a different site (Figure 4A). The site structure possessed an internanoparticle gap as narrow as 2 nm (Figure S5A, Supporting Information). The previously missed features, i.e., 1515 (xanthene ring and its attached ethylamine groups) and 616 cm−1 (xanthene and phenyl rings) showed up. Some weak features in the calculated SERS for the free R6G molecule, i.e.,

1608 (phenyl ring), 1476 (ester group attached to the phenyl ring), and 1183 cm−1 (phenyl ring), were surprisingly enhanced (trace c in Figure 4A). Meanwhile, the features at 1658, 1579, 1540, and 1213 cm−1 with the main PED distributions on the xanthene and attached groups disappeared. With the increase of the interparticle gap (Table 1S, Supporting Information), most of the features in the calculated Raman spectrum of free R6G (Row 4 in Figure S5, Supporting Information) became observable except for those at 1424 and 772 cm−1 (with PED on all groups), although the measured peak intensities somewhat varied. This is understandable because the larger the interparticle gap, the smaller the Ag nanoparticles under our preparation conditions and hence the larger the surface area of the nanoparticles. This resulted in enhanced adsorption of the molecules at hotspots that, together with the complicated plasmon modes, leads to the enhancement of the vibrational modes. In consideration of all the features observed in the Raman spectra of 10−15 M R6G, most of the intense features in the calculated Raman spectrum of free R6G were actually identified. In combination with the above-mentioned points, the observed SERS features should originate from the R6G molecule, with either the vibrational bands of the xanthene ring (and its attached groups) or the phenyl ring (and its attached groups) selectively enhanced under different experimental conditions, i.e., different substrates and different excitation lasers which together determine the LSPR, λmax. It should be pointed out that the selective enhancement of the vibrational bands with the PED over entire molecules17 was seldom observed. 3.3. Lineshape and Feature Position Analyses. The Raman spectra of the R6G on the densely packed AgNP array at concentrations of 10−8, 10−12, and 10−15 M were summarized in Figure 4A after background subtraction and spectral normalization for the purpose of line shape comparisons. In previously reported experiments, the incident excitation laser power was in the range of 1.5−0.2 mW.8,19,21 In our experiments, the employed laser power was in the range of 0.1−0.05 mW, well below 1.5 mW. To exclude the effects of the laser power and data acquisition time for the background 22706



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subtraction and normalization, the measured Raman intensity was scaled to ADU·s−1·mW−1. Upon the irradiation of a low intensity laser, the Raman signal intensity can be in proportion to the laser intensity.32 The line width (full width at half-maximum, fwhm) of the 10−15 M R6G SERS peaks (Figure 4B) could be well fitted with a Lorentzian line shape, yielding a fwhm as narrow as 7−12 cm−1. This is very similar to the features for single R6G molecules in LT-TERS.17 Meanwhile, the spectra recorded at two other sites for the same R6G concentration yielded a line width as broad as 34 cm−1, with an average of 18 cm−1 in fwhm, representing typical collective molecule adsorption behavior in the nanogaps of more than 5 nm separating neighboring AgNPs. However, these spectra simultaneously contained the Lorentzian features with a narrow line width of 7−10 cm−1 (see Figure S2, Supporting Information, for the features in the 100− 1000 cm−1 region), suggesting the single-molecule adsorption behavior in the nanogaps as well. Therefore, these spectra inferred that mixed adsorption behaviors of individual and collective molecule adsorption took place simultaneously. If the concentration increased to 10−12 and 10−8 M, typical features with a broad line width of 15−30 cm−1 fitted with the Gaussian shape were observed (Figure 4B) in the SERS spectra. The broadening of the features at such higher concentrations suggested that an ensemble effect came into play for the collective adsorption behavior of several R6G molecules in a nanogap. Moreover, the spectra for the concentrations of 10−12 and 10−8 M R6G shared common features extensively. Obviously, both the line shape and feature position of the SERS spectra were a function of the R6G concentration. In addition to the narrowing down of the line width, the features also shifted in the spectra as the R6G concentration decreased (see Figure S3, Supporting Information, in the 100− 1000 cm−1 region). The features located at 1306, 1540, and 1581 cm−1 shifted either downward or upward, but their intensities were significantly enhanced, compared with those in the averaged ensemble spectra of the R6G molecules at high concentrations (10−12 and 10−8 M). These three features had a potential energy distribution (PED) involving localized vibrations of the ethylamine moieties and xanthene (or phenyl) rings, according to the theoretical calculations by van Duyne et al.17 New features located at 1145, 1209, 1288, 1270, 1416, and 1452 cm−1 had a PED involving localized vibrations of the phenyl ring, ester group attached to the phenyl ring, the xanthene ring, or ethylamine moieties.17 Some features showed little shift in frequency in the spectra recorded at different sites for the 10−15 M R6G solution. Interestingly, features positioned at 1209 and 1540 cm−1 were also observed as shifted modes in LT-TERS,17 suggesting a similar adsorption mode on the 2D AgNP array. The unshifted features at 1608 and 1647 cm−1 were attributed to the total symmetric stretch of the CC in the xanthene ring, which are less sensitive to the local environment and absorption configurations. 3.4. Time-Evolved Fluctuation of the SERS Spectra. Spectral fluctuations frequently observed in time-evolved SERS spectra are characteristics of single-molecule adsorption behavior at hotspots.35 A scrutiny of the time-evolved SERS spectra of the 10−15 M R6G molecules collected with laser lights of 633 and 514 nm in wavelength (Figure 5) could therefore be used to study the dynamic process of the R6G molecule adsorption on the densely packed 2D AgNP array. In one sampling spot, no spectral shift or relative intensity change could be observed in the time-evolved SERS spectra (Figure

Figure 5. Time-evolved SERS spectra of the R6G in 2.4 × 10−15 M solution. (A) λex = 633 nm, Pacq = 0.1 mW, exposure time = 10 s, accumulation number = 3, in the time sequence from trace a through trace c. (B) λex = 514 nm, Pacq = 0.05 mW, exposure time = 10 s, accumulation number = 10, in the time sequence from trace a through trace j.

5A), although the absolute feature intensities indeed weakened (from trace a through trace c) as time elapsed. This experimental phenomenon in the time-evolved SERS spectra suggested a stable circumstance where the molecules stayed. Due to the hotspot heterogeneity,35 the SERS signals could only stem from a localized single molecule with a confined configuration. Moreover, the narrow line width and well-fitted Lorentzian line shape also implied the single-molecule adsorption behavior in the gap. In another sampling spot, the SERS spectra (Figure 5B) did fluctuate with time, suggesting that the molecules roamed around at the substrate surface under laser illumination (trace a through trace h in Figure 5B) until they were trapped by a hotspot and stayed in a relatively stable state (trace i through trace j in Figure 5B). Some of the spectral features could be well fitted with the Lorentzian line shape, while others could 22707



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Figure 6. Schematic growth model for the AgNP array. Blue color represents the porous Al substrate. Red color represents the Ag deposit. Colors from blue to red qualitatively indicate the height increase of the structure. Steps (a), (b), and (c) correspond to the initial deposition, preferential growth at the vertices and curved edges, and eventual development of the AgNPs at the vertices on the porous Al substrate. For details refer to the main text.

only be fitted with the Gaussian line shape, which indicated that the molecules jumped into and out of the nanogap.

However, if the average number of the R6G molecules was fewer than that of the hotspots, the chance to observe a single R6G molecule increased dramatically. 4.3. Molecule Adsorption Behavior. The size of the nanogaps was distributed over the 2−10 nm range. Statistically, about 30% nanogaps were smaller than 5 nm, 50% nanogaps were 5−9 nm, and 20% nanogaps were larger than 10 nm. According to the literature,17 the occupation area for each R6G molecule is about 2 nm2 on the Ag surface. When an aqueous R6G solution was dropped onto the AgNP array, some R6G molecules together with water molecules might fall into the nanogaps due to the capillary effect. In the nanogaps, molecule adsorption and desorption would take place rapidly until an equilibrium was established between the molecule adsorption onto the Ag surface from the solution and molecule desorption from the Ag surface back into the solution. When water evaporated, the R6G molecules around the nanogaps might diffuse into nearby nanogaps until a stable state established. As the nanogap size was comparable with that of the R6G molecule, there was the possibility that the molecule be trapped in the nanogap or chemically adsorb on the AgNPs. Chemical adsorption of the R6G molecule on the Ag surface happened by binding the N atom in ethylamine groups attached to the xanthene ring, as evidenced by the Raman feature at 230−235 cm−1 which was attributed to the Ag−N stretching mode.31 For the nanogaps of 2−5 nm in size, the narrower the nanogap, the lower the possibility that more than one R6G molecule be simultaneously trapped in one nanogap. Furthermore, surface chemical adsorption and the molecular steric hindrance effect set up a “potential barrier” for the R6G motion. As a consequence, the trapping mode was established, leading to a high stability for the time-evolved SERS spectra (Figure 5A). For nanogaps of 5−9 nm in size, there existed the possibility that several R6G molecules simultaneously adsorbed at one hotspot. In such a situation, time-dependent signal fluctuation could be observed in the time-evolved SERS spectra (Figure 5B). The intensity fluctuation in time-evolved Raman stems from the intensity change of the local electromagnetic field on the substrate and the optimized condition variation for the enhancement of a particular Raman feature37,38 due to possible hybridization of the plasmon modes37 on our 2D AgNP array. When a large number of molecules are involved, the ensembleaveraged intensity of the bands under different excitation conditions and at various hotspots was detected, which would smear out the fine SERS features. However, when only a few molecules were involved, the LSPR would come into play, which resulted in the observation of the fine SERS features, i.e., 1069, 1305, 1415, and 1453 cm−1.

4. DISCUSSION 4.1. Fabrication Method for the Closely Packed 2D AgNP Array. As mentioned above, the template-assisted electrochemical deposition was applied to fabricate the densely packed 2D AgNP array. In this approach, patterned Al foils were used as both the cathode and template in the electrochemical deposition process. To obtain the densely packed 2D AgNP array, the electrochemical deposition parameters such as deposition time, voltage, and electrolyte concentration were optimized to duplicate the periodicity of the porous Al substrate. Here, the interpore spacing (94 nm in Figure 1f) was about twice as large as the nanoparticle diameter (45 nm in Figure 2B), yielding a very small gap between neighboring AgNPs (Figure 2C). The patterned Al surface acted as a template via the so-called “protruding growth” mechanism36 at the metal cathode, as schematically shown by Figure 6. Steps (a), (b), and (c) correspond to the initial stage, developing stage, and late stage of the growth procedure. Under the experimental conditions, initial Ag deposition (step a in Figure 6) could simultaneously take place at vertices, curved edges, and the bowl bottom of the porous Al substrate (cf. Figure 1f). The difference in the local electric field enhancements at the vertices, curved edges, and the nanopit bottoms amd the Ag growth rate was in the order, vertices > curved edges > nanopit bottoms (step b in Figure 6). As a consequence, AgNPs eventually developed at the vertices (step c in Figure 6). Careful control of the experimental parameters allowed the formation and tuning of the small gaps between the AgNPs on purpose. 4.2. Number of Molecules Sensed. One could estimate the average number of molecules within the laser spot by carefully calculating the coverage of the AgNP array after electrochemical deposition, probing volume and concentration of the R6G solution. The average number of molecules in the detection region for 2.4 × 10−8, 2.4 × 10−12, and 2.4 × 10−15 M R6G (in solutions) was about 105, 10, and 10−2 molecules, correspondingly. Given the total surface area of the AgNPs in the laser spot and the size of the R6G molecule,17 the total number of the R6G molecules at full coverage was 106 molecules/cm2 under our experimental conditions. However, the number of the R6G molecules absorbed at the effective hotspots was limited by the number of the nanogaps between neighboring AgNPs. The latter was estimated to be about 290 according to the periodicity (94 nm) of the AgNP array and the laser spot size (1 μm). This meant that when the number of the R6G molecules exceeded 290 an ensemble-averaged feature in the SERS spectra31 would be observed as shown in Figure 4. 22708



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Figure 7. (A) FESEM image and (B) corresponding schematic model showing the formation of a 2D AgNPT lattice. The black rectangle in (B) indicates a unit cell of the 2D rectangular AgNPT lattice, being about 180 nm × 207 nm in size and containing two AgNPTs. The AgNPTs in neighboring rows in the sample point in opposite directions.

5. CONCLUSIONS In summary, we have successfully fabricated a densely packed 2D AgNPT array by the electrochemical deposition on the closely packed porous Al template. This 2D structure dramatically enhanced the signal intensity in the SERS spectra and achieved a very high sensitivity of the R6G molecules in solution with a concentration as low as 10−15 M. Both line shape and feature position in the SERS spectra were sensitive to the solution concentration. At a low solution concentration, single-molecule adsorption behavior came into play in the SERS spectra. This was further supported by the analysis of the number of molecules in the probe region. Time-evolved SERS spectra showed a high signal stability at one spot, characteristic of the single-molecule adsorption behavior, and an intense signal fluctuation in the SERS spectra at the other spot of the same sample surface, indicative of multimolecule adsorption behavior. Both substantiated that there was coexistence of single-molecule adsorption behavior in the 2−5 nm nanogaps and multiple molecule adsorption behaviors in the 5−9 nm nanogaps. The detection limit down to the single-molecule level on our prepared sample mainly stemmed from the formation of the 2D “hotspot lattice” of the AgNPT array. Such a highly sensitive SERS structure might have wide applications in chemical detection, environmental analysis, medical diagnosis, drug sensing, and so on and so forth.

The stability is likely relating to the narrow gap in the R6G size (see, for example, Figure 2D) which sets up a “potential barrier” preventing the trapped R6G molecules from jumping out of the gap during the water evaporation process. Moreover, the N−Ag vibrational feature at 230−237 cm−1 (Figure 5A) indicated that the R6G molecules were chemically bonded to the substrate surface. If a molecule was not trapped in the gap, it would diffuse upon laser illumination which intensified its thermal motion on the surface until it was trapped in a small gap so that the molecule was “frozen” in place. As such, “fluctuation-stable” time-evolved spectra were obtained (Figure 5B and Figure S4, Supporting Information). This would account for our experimentally observed spectral and intensity fluctuation as time evolved, in contrast to the ensembleaveraged broad Raman signals in normal room-temperature SERS measurements. 4.4. Origin of the High Sensitivity of the 2D AgNP Array. There are a couple of reasons responsible for the high sensitivity down to the single-molecule limit for our prepared close-packed AgNP array. First, both charge transfer and huge electromagnetism played a key role. Second, the formation of the AgNP−R6G−AgNP junctions at the nanogaps of 2−5 nm further contributed to the high sensitivity. Third, each AgNP in our sample was surrounded by three AgNPs so that tetramer hotspots were formed (Figure 7A). According to the model (Figure 7B), the rectangular unit cell of the AgNP tetramer was measured to be about 280 nm × 207 nm, containing two AgNP tetramers. A previous study has shown that a trimer hotspot enhances the local electromagnetic field more effectively than a dimer hotspot.38,39 One would reasonably expect that a much better enhancement factor existed for the AgNP tetramer containing more than one trimer. Moreover, a further enhancement originated from the collective couplings of the AgNP tetramers. Fourth, the closely packed AgNP structure presented a high density of the hotspots. Finally, the tetramer hotspots formed the patterned structure extending to tens of micrometers in area, yielding a 2D-crystal-like structure which augmented the molecule adsorption probability at these hotpots. In brief, this type of 2D “hotspot lattice” structure presents three advantages: (1) The formed AgNP tetramers have a much higher SERS enhancement than do the AgNP dimers and trimers. (2) The AgNP tetramers form a 2D “hotspot matrix” that generates a collective enhancement effect.28 (3) The AgNP tetramers offer dense surface sites for the adsorption of the R6G molecules. All these factors led to that our prepared closely packed 2D AgNP array possessed a very high sensitivity for the detection of the R6G down to the single-molecule limit.

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ASSOCIATED CONTENT

S Supporting Information *

This part includes supplementary morphologies of the nanostructure, spectral comparison, and peak fitting results of the silver SERS substrates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was jointly supported by NSFC (51121091, 21133001, 21261130090, 51121091, 21228301) and MOST (2011CB808702), China. Partial support from the Singapore NRF CREATE-SPURc project is also acknowledged. 22709



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Two-Dimensional Ag Nanoparticle Tetramer Array for Surface

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