Fabrication of Large-Area, High-Enhancement SERS Substrates with

Article pubs.acs.org/JPCC

Fabrication of Large-Area, High-Enhancement SERS Substrates with Tunable Interparticle Spacing and Application in Identifying Microorganisms at the Single Cell Level Jing Chen,† Bo Shen,† Gaowu Qin,*,† Xianwei Hu,‡ Lihua Qian,§ Zhaowen Wang,‡ Song Li,† Yuping Ren,† and Liang Zuo† †

Key Laboratory for Anisotropy and Texture of Materials, Ministry of Education, Northeastern University, Shenyang 110819, China College of Materials and Metallurgy, Northeastern University, Shenyang 110819, China § School of Physics, Huazhong University of Science and Technology, Wuhan 430074, China ‡

S Supporting Information *

ABSTRACT: The interparticle spacing of the surface-enhanced Raman scattering (SERS) substrate has a strong relationship with its enhancement factor (EF). How to precisely adjust the interparticle gap and generate SERS substrates with excellent quality and high reliability by a facile way is still a challenge. Here, we propose a convenient and environmentally friendly method to synthesize large-area Ag SERS substrates composed of either monodisperse nanoparticles (NPs), NP-linked nanowires (NWs), NW-weaved mesoporous membrane, or NP-aggregates by simply controlling the pH value in alkaline glucose solution, and their SERS enhancements have been evaluated. In addition, the EF of the Au NW-weaved film substrate prepared by our method is one order higher than that well-known dealloyed Au nanoporous substrate. Finally, the SERS spectrum of yeast at the single cell level is successfully acquired by using the highest EF substrate composed of monodisperse Ag NPs (∼8.24 × 107) in this work at a very low laser power (0.17 mW).

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INTRODUCTION Surface-enhanced Raman scattering (SERS) has been paid increasing attention for several decades due to its marvelous enhancement and excellent sensitivity and selectivity among the vibrational spectrum when the analyte is close to a nanoscale, rough metal surface.1−5 As the unique local plasma resonance of noble metal nanomaterials, they have usually been employed for producing substrates that can be used in SERS to fulfill the detection of chemical and biological molecules.6−9 A great breakthrough in SERS was the achievement of single-molecule detection in 1997.10,11 However, it is still a challenge to produce portable and cost-effective SERS sensors so far, and one of the reasons is the difficulty in fabrication of reliable and reproducible SERS substrates. Currently, SERS enhancement is generally considered to arise from the electromagnetic effect and the chemical effect, the former of which is normally deemed to play a vital role in enhancing Raman signal. The maximum enhancement occurs in the hot spots, which refer to the regions usually less than 10 nm12 between metallic nanoparticle (NP) pairs, where surface plasmon of the metal NPs can be coupled to each other to produce more intense electromagnetic fields.13 Hence, how to prepare reliable SERS substrates with a large number of hot spots is of great importance in both fundamental and practical senses. Hong et al. fabricated two-dimensionally macroporous Ag films with adjustable periodic distances using nanosphere © 2012 American Chemical Society

lithography on the solution surface combined with interfacial reactions, which showed an enhancement factor (EF) as high as 107.14 Mulvihill et al. reported a chemical method for preparation of silver nanocrystals with new shapes by using a highly anisotropic etching process, and these new etched silver particles can serve as highly sensitive SERS substrates.15 Alexander and co-workers reported an ingenious method by which the gap between two gold nanorods can be tuned by varying the strain applied to a stretchable elastomeric silicone rubber substrate. Thus, the distance dependence of Raman scattering can be tracked under a single nanorod dimer.13 More recently, the Zhang group found a synergistically enhanced SERS signal of Shewanella oneidensis when Ag NPs and Ag nanowires (NWs) were used together, contributed by the dramatically enhanced electromagnetic field due to the presence of two different metallic nanostructures.16 However, it is worth noting that nowadays most of the preparation methods involve many cumbersome procedures when it comes to large area SERS substrates. Besides, how to accurately adjust the spacing between hot spots is always of great importance for SERS enhancement. Herein, we introduce a simple and green method for producing large area SERS substrates, which can provide uniform and Received: October 22, 2011 Revised: December 31, 2011 Published: January 9, 2012 3320

dx.doi.org/10.1021/jp210147c | J. Phys. Chem. C 2012, 116, 3320−3328

The Journal of Physical Chemistry C

Article

way, an Au SERS substrate composed of Au NP-linked NWs was also fabricated. To obtain the NPGL, a piece of 100 nm thick, 12 carat white gold leaf (Ag/Au, 1:1 ratio by weight) was dealloyed by floating on concentrated nitric acid (65%∼68%) for 1 h and then rinsed with ultrapure water carefully several times. Finally, the obtained NPGL was transferred onto a piece of glass slide for the SERS measurement. Sample Preparation for Measuring Substrates Enhancement Capability. The above four different constitutions of Ag SERS substrates, Au SERS substrate, and the NPGL substrate were dipped into 0.01 M Py aqueous solution for 30 min each; then, they were immediately transferred to get SERS spectra. Normal Raman spectroscopy of 0.1 M Py aqueous solution was also acquired. Preparation of Yeast Cells Sample. The yeast cells were grown in 10 mL of YPD culture media (10 g of yeast extract, 20 g of peptone, 20 g of dextrose, and 1000 mL of sterile water), and they were incubated by oscillation at 28 °C for 24 h in ambient conditions. Then, they were collected by centrifugation at 8500g for 6 min. The supernatant was discarded. The bottom yeast cells were washed by sterile water three times and resuspended in 0.5 mL of water. About 10 μL of the yeast cell suspension was dripped on to the Ag SERS substrate with the highest EF and on a piece of glass slide, respectively. Then, they were dried at room temperature. Characterization. Transmission electron microscopy (TEM) observations of the samples were performed by using JEM-2100F, JEOL, with the acceleration voltage of 200 kV. For TEM sample preparation, 5 μL of the solution was dropped onto standard carbon coated Cu grids, respectively. Field emission scanning electron microscopy (FE-SEM) was carried out on JSM-7001F, JEOL, with the operation voltage of 15 kV. The X-ray diffraction (XRD) pattern was studied by an X’Pert Pro diffractometer (PANalytical Co., Holland) with Cu Kα radiation (40 kV and 40 mA). The ultraviolet−visible-nearinfrared (UV−vis-NIR) absorption spectra were recorded by 756PC UV−vis Spectrophotometer, Shanghai Sunny Hengping Scientific Instrument Co., Ltd. The roughness factors of SERS substrates were obtained by using Dektak 150 Surface Profiler, Veeco, and follow-up calculation. The confocal laser scanning microscope was carried out on Olympus LEXT OLS3100. All SERS and bulk spectra were obtained using a confocal Horiba Jobin Yvon LabRAM HR800 Raman spectrometer equipped with a He−Ne laser (632.8 nm) and a 50× long workingdistance objective (10.6 mm) to focus the laser onto the sample surface and to collect the scattered light from samples. The incident laser power was attenuated to 0.17 mW for most of the SERS measurements in this article except for the NPGL substrate with 8.5 mW, and the laser power for the normal Raman spectra acquisition of 0.1 M bulk Py solution was set as 17 mW. The laser spot was 1.54 μm in diameter, and the pinhole used in this experiment was set as 50 μm. The frequency of the Raman instrument was calibrated by referring to a silicon wafer at the vibrational band of 520 cm−1. Each spectrum was collected with a 15 s exposure time and averaged by 5 scans. All the measurements were carried out in ambient conditions.

sufficient enhancement. A series of Ag and Au NPs or NWs can be easily fabricated in alkaline glucose solution at room temperature by simply controlling the pH value of solution, and thus, monodisperse Ag and Au NPs or their nanoporous films are finally obtained.17 In this approach, the interparticle spacing of the NPs can be tuned handily and precisely, while keeping the same size and shape of the NPs, so the EFs of different SERS substrates can be investigated by only changing one variable, which is the aim of many fabrication methods being explored. At the same time, large area substrates prepared here have many hot spots presenting in the long term stability. What’s more, the glucose acts as both a reduction and capping agent, and the water is the solvent, all of which conform to the green chemistry principles. In our technique, four kinds of Ag SERS substrates (monodisperse Ag NPs, NWs, mesoporous films, and aggregates), which were all reduced by D-glucose solution with different pH values, were applied to detect their SERS enhancements. For comparison, a 100 nm thick nanoporous gold leaf (NPGL)18 and a piece of Au NW-weaved mesoporous film prepared in glucose solution at the initial pH of 12.017 were also applied to test their SERS EFs. Pyridine (Py) was selected as the model molecule to measure substrates' EFs, and the theoretical analysis regarding the different enhancement capabilities and the underlying enhancement mechanism were finally clarified. To explore their application in biological sensor, one microorganism at the single cell level was also selected as the probe.19−26 A yeast cell with the external surface composed of mannoproteins, which is considered to be one of the most deeply researched eukaryotic organisms, was selected for the SERS measurement in this work.

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EXPERIMENTAL SECTION Reagents and Materials. AgNO 3 , HAuCl 4·4H 2 O, D-(+)-glucose, NaOH, pyridine, and HNO3 (65%-68%) were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd., and all of which were used without further purification. Cellulose acetate microfiltration membranes (CAMMs) were purchased from Xinya Purifier Devices Factory, Shanghai, China. Ultrapure water (18.2 MΩ·cm) obtained from an ultrafilter system (CSR-1-10, Aisitaike Technology Development Co., Ltd., Beijing, China) was used throughout the experiments. Synthesis of Ag NPs, NWs, Mesoporous Films, and Aggregates. In a typical procedure, 108 mg of D-(+)-glucose was added into 20 mL of ultrapure water to get a 0.03 M glucose aqueous solution, then 0.1 and/or 1 M NaOH aqueous solution was added dropwise to adjust the pH values (called the initial pH value of glucose solution in this article, unless otherwise specified) with stirring to make them homogeneous. Finally, 80 μL of 0.05 M AgNO3 aqueous solution was dropped quickly into the above basic glucose solution, and the mixed solution was shaken promptly, then kept at room temperature for several hours. Preparation of the SERS Substrates. When AgNO3 aqueous solution is added into the alkaline glucose solution, Ag+ would be reduced to Ag NPs. Afterward, depending on the various pH values, four kinds of Ag products would be produced; that is, stable and monodisperse Ag NPs, short Ag NWs, long Ag NWs that will self-support to form nanoporous film, and aggregates of unstable Ag NPs. Then, four kinds of Ag SERS substrates (monodisperse NPs, NP-linked nanowires, NWs interlaced self-supporting mesoporous membrane, and aggregates) would be fabricated by depositing the above four kinds of Ag products onto CAMMs, respectively. In the same

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RESULTS AND DISCUSSION Preparation and Characterization of Four Kinds of Ag SERS Subatrates. The initial pH value has a significant influence on the stability and linking behavior of Ag NPs. When 80 μL of 0.05 M AgNO3 solution was added into 20 mL of 3321



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Figure 1. (a) XRD pattern of Ag NPs reduced at the pH value of 10.40. (b) UV−vis-NIR absorption spectra of Ag NPs reduced under different initial pH values (one minute after preparation). (c) The evolution of UV−vis-NIR absorption spectra with time at pH = 10.40. (d) FE-SEM image of SERS substrate composed of Ag NPs reduced at the initial pH value of 10.40, inserted is the corresponding TEM image.

0.03 M glucose alkaline solution, four kinds of Ag products would be formed depending on the various initial pH values. First, monodisperse Ag NPs stabilized by glucose are produced when the initial pH value is in the range of 9.30−11.20. The XRD pattern in Figure 1a verifies that Ag NPs are of facecentered cubic structure. The UV−vis-NIR absorption spectra of Ag NPs produced under different pH values within this pH range are shown in Figure 1b, and these spectra were collected as soon as the reaction occurred. The absorption peak is at ∼416 nm, and the Ag NP absorption intensity increases with rising pH value, implying more Ag NPs are reduced at stronger solution basicity within this pH range. The evolution of UV−vis-NIR absorption spectra with time at pH = 10.40 are shown in Figure 1c, and these spectra do not show distinct difference even after 12 h reaction, indicating the stability of Ag NPs reduced in this pH range. The FE-SEM image of Ag NP SERS substrate prepared at pH = 10.40 (Figure 1d) shows that the NPs are close-packed, with a mean grain size of about 40−50 nm. The majority of the NP pairs are with small interparticle distance, which is highly beneficial for the SERS enhancement. TEM image of Ag NPs reduced at the initial pH of 10.40 (inset of Figure 1d) indicates the monodispersity of Ag NPs formed in this pH range. Second, when the initial pH value of the glucose solution is in the range of 11.30−12.60, Ag NPs tend to weld each other to form Ag NWs. However, there are two kinds of Ag NWs that can be observed depending on different pH in this range. That is, in the lower pH range of 11.30−11.70, short Ag NWs, which consisted of three or four Ag NPs linking, are formed, and in the higher pH range of 11.80−12.60, longer Ag NWs are

formed. Then, the NWs randomly intertwine each other into a self-supporting network just like the mesoporous Ag sponge but with different pore sizes depending on the length of Ag NWs in which short NWs pile up more closely and result in smaller pore sizes than long NWs do. Finally, the cross-linked NWs will precipitate out as a well whole thin membrane consisting of a continuous nanostructure but with different precipitated time, which a higher pH value causes quicker sediment. To explore the underlying interconnected behavior of Ag NPs in the pH range of 11.30−12.60, UV−vis-NIR absorption spectra at a given pH value but with different reaction time were measured. Figure 2a is the evolution of UV−vis-NIR absorption spectra with time for the reaction solution at the initial pH of 11.70 representing the pH range of 11.30−11.70, in which short Ag NWs form. Here, it can be observed that an intensity decrement at 416 nm with time, together with an increased absorption intensity in the NIR region. The peak at 416 nm represents the surface plasmon absorption band of Ag NPs, while the NIR region represents mutual surface plasmon absorption bands of Ag NWs along different longitudinal orientations. The decrease in adsorption intensity at 416 nm, together with an increasing in the NIR region in Figure 2a indicate Ag NPs have welded each other to form Ag NWs.17,27 Both FE-SEM (Figure 2b) and TEM images (inset of Figure 2b) verify that Ag NWs indeed form at pH = 11.70, and most of the NWs are composed of less than five welded Ag NPs. Similarly, the evolution of UV−vis-NIR absorption spectra with time at pH = 12.30 representing the pH range of 11.80− 12.60, in which long Ag NWs form, are also shown in Figure 3a. 3322



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Figure 2. (a) Evolution of UV−vis-NIR absorption spectra with time at pH = 11.70, which correspond to the transformation from monodisperse NPs to short NWs. (b) FE-SEM image of SERS substrate composed of short Ag NWs forming at the initial pH of 11.70, inserted is the corresponding TEM image.

Figure 3. (a) Evolution of UV−vis-NIR absorption spectra with time at pH = 12.30, which correspond to the transformation from monodisperse NPs to long NWs. (b) FE-SEM image of SERS substrate composed of long Ag NWs forming at the initial pH of 12.30, inserted is the corresponding TEM image. (c-f) Visual observation of the curling process of the continuous, self-supporting and mesoporous Ag film forming at the initial pH of 12.30.

continuous film composed of random assembly of long Ag NWs forming at pH = 12.30, and the inset of Figure 3b is the TEM image of long Ag NWs forming at the initial pH of 12.30. In addition, we should note that this kind of membrane exhibits a much looser nanostructure and larger pore sizes than the films in Figure 2b do, and both of the pore sizes with micrometer scales in the above FE-SEM images result from the leaching procedure in SERS substrate preparation. Figure 3c−f reveals the visual observation of the curling process of this

Same as Figure 2a, the extinction of the peak at 416 nm, together with a lifting in the NIR region in Figure 3a, also reveal the linking behavior of Ag NPs, but we should note that there are some differences between Figure 2a and Figure 3a. In Figure 3a, linking behavior occurs as soon as Ag NPs have been reduced, which can be reflected from the NIR region lifting of the 1 min curve. Besides, the 5 h curve in Figure 3a is a flat line, which indicates a quicker linking and sediment behavior. The FE-SEM image shown in Figure 3b reveals a self-supporting, 3323



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catalyzed by acid. According to the different configuration of the formed hemiacetal group, the cyclic chair form is classified as α-D-(+)-glucopyranose and β-D-(+)-glucopyranose, with the final equilibrium percentage of 36.4% and 63.6%, respectively. This conversion process is presented in Scheme S1, Supporting Information. It is the aldehyde group in D-(+)-glucose to play the part of reduction, but in glucose aqueous solution, the existent abundant H+ would catalyze the open chain form of D-(+)-glucose to transform to the cyclic chair form, and in cyclic chair form, the carbonyl would be protected, and D-(+)-glucose could not play the role of reduction. When the NaOH aqueous solution is added to make the glucose solution weakly basic, the open chain form of D-(+)-glucose is gradually formed because of the decreasing concentration of H+, thus the aldehyde group will be exposed, and Ag+ is reduced. Along with the alkalinity increasing, more aldehyde groups are unfolded, so more Ag NPs are produced, which explains the phenomenon in Figure 1b that Ag NP absorption intensity increases along with pH value rising. Similar to the finding confirmed by Sylvestre,28 there may be also an equilibrium between −OH and O− on the synthesized Ag NPs surface, depending on the pH. In detail, when pH is below the pK value of the hydroxylated surface of Ag NPs, −OH groups are dominant for the Ag NPs protection; on the contrary, O− groups are in a dominant position.28 Besides, glucose would be oxidized to gluconic acid by reducing Ag+ or by the slow self-oxidization with the aid of O2. Thus, the forming mechanism of the above four kinds of Ag SERS substrates can be expected as seen in Scheme 1. First, in a weakly alkaline condition, such as pH 9.30−11.20, Ag NPs would be capped with the produced −COOH due to polarity17 and −OH, thus a stable and dispersive colloid solution would form. Then, with pH value increasing, the abundant OH− would inevitably react with −COOH to form −COO−, and at the

continuous, self-supporting and mesoporous Ag film precipitated out at pH = 12.30. Finally, at very high initial pH (>12.60), Ag NPs would attach each other closely from every direction, thus resulting in aggregates, together with the coarsened Ag NWs. A TEM image exhibiting the structure of the sample prepared at pH = 12.80 is shown in Figure 4.

Figure 4. TEM image of Ag-NP aggregates together with some coarsened Ag NWs forming at the initial pH of 12.80.

Clarification of the Forming Mechanism of the Above Four Kinds of Ag SERS Substrates. The results presented above show a high dependence of linking behavior of the initially reduced Ag NPs on the initial pH values. Then, we will explain this dependence from the structure of glucose. D-(+)-Glucose is the aldohexose containing carbonyl and hydroxyl, with an open chain form. Nevertheless, the majority of D-(+)-glucose in aqueous solution exist in the form of intramolecular hemiacetal through the reaction between the aldehyde group on carbon-1 and the hydroxyl group on carbon-5, which is

Scheme 1. Formation of the above Four Kinds of Ag SERS Substrates

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same time, Ag−O− groups would take the lead. Hence, in a strong alkaline surrounding, Ag NPs surface would be covered by −COO−, −OH, and −O−. When a Ag NP meet with another adjacent Ag NP, the strong electrostatic repulsive force would make neighboring capping ligands instantaneously stripped off, which generates some naked areas on the surface of Ag NPs. To decrease the surface free energy, these naked areas would connect to each other by Brownian motion,17 thus short Ag NWs are formed in the pH range of 11.30−11.70, and the stronger the alkalinity is, the more −COO− and −O− groups that would be generated. So, with pH value further rising to 11.80−12.60, more −COO− and −O− groups would wrap an individual Ag NP, leading to bigger odds of connection, thus longer Ag NWs form. Finally, at very high initial pH (>12.60), aggregates together with some coarsened Ag NWs are observed. It can be suggested that at this high range of pH values, large amounts of D-(+)-glucose with an open chain form are rapidly formed, and the abundant aldehyde groups are exposed to render the glucose solution as having a strong reduction ability. Once AgNO3 solution is added, plenty of Ag NPs form in an instantaneous moment and tend to aggregate to reduce surface energy before their surfaces are well capped by −COO− and −O− groups all around.17 Therefore, the much larger sized Ag aggregates precipitate from the solution in a short time. EF Measurements for Different SERS Substrates. To explore the SERS enhancement mechanism, the above four kinds of Ag SERS substrates that are monodisperse NPs, short NWs, long NWs, and aggregates prepared at the initial pH value of 10.40, 11.70, 12.30, and 12.80 were selected for SERS measurement. Py was employed as the probing molecule because of its large Raman scattering cross-section and wellidentified Raman spectrum. The SERS spectra were acquired (Figure 5) after dipping the above four substrates in 0.01 M Py aqueous solution for 30 min.

in the following by referring to the calculation procedure in ref 29. The most widely used definition for EF (G) is30

I N G = surf × bulk Ibulk Nsurf

(1)

where Isurf and Ibulk are the integrated intensities of the ring breathing band of Py adsorbed on Ag or Au SERS substrates and in the bulk solution, respectively. Nsurf and Nbulk are the corresponding number of Py molecules on the SERS active substrates and in the bulk solution effectively illuminated by the laser beam, respectively. Provided that Py molecules were in monolayer adsorption,

RA (2) σ where R is the roughness factor of a substrate, A is the area of the laser focal spot, and σ is the surface area occupied by a Py molecule. For Py, there are usually two adsorption configurations: the flat adsorption and the vertical adsorption. According to the literature,31,32 it seems quite possible that in our experiment, Py molecules are in the vertical adsorption geometry. For this vertical orientation, the value of σ can be estimated to be 0.27 nm2 33 and 0.30 nm2 34 for Py adsorbed at Au and Ag films, respectively. Nsurf =

Nbulk = AhcNA

(3)

where c is the concentration of Py, here, c = 0.1 M, NA is the Avogadro constant, and h is the confocal depth of the laser. With eqs 2 and 3, eq 1 can be rearranged as

G=

hcNA σIsurf RIbulk

(4)

Calculation of Confocal Depth h. In the light of the model assumed by Cai et al.,29 the Raman intensity-depth profile of the 520.6 cm−1 band for a silicon wafer is made (Figure S1, Supporting Information). We considered that the Raman signals that are acquired in the region of deviating the ideally focused plane more than 50 μm (|Z|>50 μm) are negligible. So h is calculated to be 13 μm. Calculation of Roughness Factor R. The roughness factor R can be defined as the ratio of the actual area of a rough surface to the geometric projected area. In this work, R values were measured by using a surface profiler. The simulated surface image of the substrate composed of close-packed Ag NPs prepared at the initial pH value of 10.40 is given in Figure S2a, Supporting Information, and its corresponding roughness factor R is 1.048 after a series of calculations. Calculation of Isurf/Ibulk. The Raman spectrum of 0.1 M Py bulk solution, together with 0.01 M Py SERS spectra acquired from the substrate composed of monodisperse Ag NPs prepared at the initial pH of 10.40, are presented in Figure 6. The integrated intensities of the symmetrical ring breathing bands for Pysol,a (1008 cm−1) and Pyads,b (1012 cm−1) are 153 and 56 240 cps, respectively. In eq 4, Isurf/Ibulk should only be calculated when Isurf and Ibulk are obtained in the absolutely identical measurement conditions. However, considering the various enhancement capability of substrates, spectra were acquired under different laser power and with different concentration of Py solution. For example, the normal Raman spectrum in Figure 6a was acquired from a 0.1 M Py solution under 17 mW laser power,

Figure 5. SERS spectra of 0.01 M Py acquired from four kinds of Ag SERS substrates prepared at different initial pH values. Excitation wavelength, 632.8 nm; laser power, 0.17 mW.

Figure 5 shows that the SERS substrate with a close-packed monodisperse Ag NP surface topography, which was prepared at the initial pH value of 10.40, owns the highest SERS enhancement capability. With the initial pH value rising, the SERS intensity of Py decreases heavily, and the SERS intensity at 1012 cm−1 and 1040 cm−1 obtained from the substrate prepared at pH = 10.40 is about ∼100 times higher than that prepared at pH = 12.80. In order for better understanding the correlation between the substrate feature and SERS effect, the EFs of some substrates prepared in this work were figured out 3325



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of 10.40 owns the highest enhancement; next is the short Ag NWs prepared at the initial pH of 11.70; the third is the long Ag NWs prepared at the initial pH of 12.30; and the substrate with the constitution of aggregates has the lowest enhancement. This result might result from the intrinsically different microstructures of each substrate. There is much chance that the substrate comprising close-packed monodisperse Ag NPs has the most NP pairs with a spacing less than 10 nm, thus it owns the most hot spots, where the highest enhancement occurs. Naturally, this kind of substrate deserves an EF as high as ∼107. Then, when the substrates are composed of interlaced Ag NWs, the interparticle distance is enlarged, and the number of most suitably enhanced surroundings reduces. Therefore, the enhancement capabilities are lower, and the longer the Ag NWs are, the larger the interparticle spacing will tend to be, so the enhancement of substrate with long Ag NWs is lower than that of the substrate with short Ag NWs. The surface topographies of these three kinds of substrates are provided in Figures 1d, 2b, and 3b, and the different interparticle gaps reflected by these figures are in agreement with our speculation. However, when the substrate is composed of Ag NPs aggregates, each large aggregate comprises many Ag NPs attached to each other closely, together with some coarsened Ag NWs, which would lead to a further decrease of hot spots; thus, the enhancement capability of this substrate is the lowest in these four kinds of Ag substrates. The SERS enhancement capability of the Au NW-weaved mesoporous film is superior to that of NPGL, probably because of two factors. First, NPGL grains are single-crystal NW networks18 on the tens of micrometers scale. In contrast, the nature of the Au NW-weaved nanoporous film prepared in glucose solution at the initial pH of 12.0 is of polycrystal.17 Thus, more Py molecules would be adsorbed on the spots such as grain boundaries in polycrystal, which would lead to a higher Raman signal intensity. Second, the structure of Au NW-weaved mesoporous film is less ordered than the structure of the NPGL substrate. The distribution of the interparticle spacing of the NPGL substrate is mostly focused at about 20 nm, which is usually beyond the most suitable electromagnetic field enhancement gap, as shown in Figure 7a. In contrast, the interparticle spacing of the Au NW-weaved mesoporous film substrate varies from several to hundreds of nanometers (Figure 7b), and thus, there would exist more hot spots. SERS Measurement of Yeast at the Single Cell Level. Figure 8a is the normal Raman spectrum of a yeast cell deposited on a glass substrate. No Raman scattering peaks can be detected because of the strong fluorescence background interference from cell and low detection sensitivity. By using the highest enhancement substrate in this work, which consisted of close-packed monodisperse Ag NPs, the SERS spectrum of a single yeast cell can be acquired (Figure 8b). In this spectrum, all vibrational bands in Figure 8b, which delivered intrinsic surface chemical information of the yeast cell, are closely related to the peaks previously reported by Li.36 Tentative assignment of the peaks from Figure 8b are listed below. The peaks at 1587, 1411, 1341, and 1230 cm−1 are typical Raman signatures of Phe and Tyr, α-amino acids, adenine and guanine, and amide III,

Figure 6. (a) Normal Raman spectrum of 0.1 M Py bulk solution. Laser power: 17 mW. (b) SERS spectrum of 0.01 M Py acquired from the substrate composed of monodisperse Ag NPs prepared at the initial pH of 10.40. Laser power: 0.17 mW.

while the SERS spectrum in Figure 6b was acquired from a 0.01 M Py solution under 0.17 mW laser power. So, Isurf/Ibulk can not be calculated right now. However, the Isurf and Ibulk values obtained from different experimental parameters can be transformed to the values obtained from identical experimental parameters by the following equation:35

φ k = φoSkNHL4π sin 2(α /2) where ϕk is the flux of Raman scattering light, which is collected in the direction of being vertical to the incident laser by the focus lens, ϕo is the flux of incident laser irradiating onto the samples, Sk is the coefficient of Raman scattering, N is the number of molecules in unit volume, H is the active volume for samples, L is a coefficient affected by refractive index and field effect, and α is the semiangle of Raman scattering in the direction of the focus lens. In our measurement, Sk, H, L, and α are invariable, then ϕo and N are directly proportional to ϕk. So, according to this equation, the Isurf and Ibulk values can be transformed to the values taken from the same measurement conditions, then Isurf/Ibulk can be figured out. Finally, the G value corresponding to the substrate composed of monodisperse Ag NPs prepared at the initial pH of 10.40 can be calculated to be 8.24 × 107. In addition, the EFs of the substrate composed of Au NWweaved mesoporous film prepared at the initial pH value of 12.0 and the substrate of NPGL were also can be figured out to be 4.72 × 105 and 1.22 × 104, respectively. Their simulated surface images and 0.01 M Py SERS spectra acquired from these two kinds of substrates are shown in Figures S2b,c and S3 (Supporting Information), respectively. All the variables in eq 4 related to the G values of the above three kinds of SERS substrates are summarized in Table 1. Meanwhile, it should be pointed out that the EFs of the above SERS substrates involved no contributions from the resonance effect. To avoid the nonuniformity of the substrate, each spectrum was an average result of three to five measurements from different sites. Enhancement Mechanism Analysis for Various SERS Substrates. From Figure 5, it shows that the substrate composed of monodisperse Ag NPs prepared at the initial pH

Table 1. Variables in Eq 4 and EF (G) Values of the above Three Kinds of SERS Substrates substrate monodisperse Ag NPs Au NW-weaved mesoporous film nanoporous gold leaf

h (μm) 13

c(mol/L)

σ (nm2)

R

Isurf/Ibulk

G

0.1

0.3034 0.2733 0.2733

1.048 1.052 1.009

3.68 × 105 2.35 × 103 58.2

8.24 × 107 4.72 × 105 1.22 × 104

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for a single yeast cell are not exactly the same as the reported spectra in ref 36, and the reason for this may be from the different SERS substrate materials and preparation methods, which result in various adhesion and binding sites between substrate and cell ektexine. The SERS spectra of a single yeast cell is successfully obtained by 632.8 nm laser irradiation with a very low laser power (0.17 mW), which further indicates the effectiveness of our substrate. Figure 8c,d is the confocal laser image of yeast cells deposited on glass substrate and monodisperse Ag NPs SERS substrate, respectively. The yeast cell in the red circle represents the single cell that is measured. In addition, the durability of our SERS substrate has also been tested. After keeping the sample (SERS substrate with yeast cells deposited on it) of Figure 8d for one week, the SERS spectrum of a single yeast cell in Figure 8d was successfully acquired again, as shown in Figure S4, Supporting Information, which suggested high durability and reproducibility of our SERS substrate. These results indicate the potential application of this pH-mediated, interparticle distance-tunable SERS substrate fabrication technique. What’s more, the results also suggest that the substrate composed of monodisperse Ag NPs as a reliable and high sensitivity substrate is qualified for other specific molecules and cells detection in food, environmental, medical, and biological fields.

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CONCLUSIONS In summary, a handy, cost-effective, environmentally benign and template-free method has been employed in this work to prepare SERS substrates composed of either monodisperse NPs, NP-linked nanowires, NWs interlaced self-supporting mesoporous membrane, or normal NP aggregates of Ag. The geometrical features and interparticle distance of the above SERS substrates are well controlled by adjusting the initial pH values of a glucose solution precisely, and their formation mechanisms have been thoroughly clarified. SERS enhancements of these Ag substrates, together with another two types of nanoporous Au substrates, are evaluated by selecting pyridine as the model molecule, and the EF of the Au NW-weaved film substrate prepared by our method is one order higher than that of well-known dealloyed Au nanoporous substrate. Finally, the SERS substrate composed of monodisperse Ag NPs is confirmed to have the highest enhancement factor (∼8.24 × 107), and the SERS spectrum of yeast at the single cell level has been successfully acquired by using this SERS substrate. These results demonstrate the potential application of this pH-mediated SERS substrate fabrication technique, and the SERS substrate prepared in this work is promising for future trace detection of bacteria and organic contaminants.

Figure 7. (a) TEM image of the NPGL sample dealloyed for 1 h in concentrated nitric acid (65−68%). (b) FE-SEM image of the substrate, which consisted of an Au NW-weaved mesoporous film forming at the initial pH of 12.0.

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ASSOCIATED CONTENT

* Supporting Information S

Figure 8. (a) Normal Raman spectrum for a yeast cell after the subtraction of substrate interference. Excitation wavelength, 632.8 nm; laser power, 17 mW. (b) SERS spectrum of a single yeast cell after the subtraction of substrate interference. Excitation wavelength, 632.8 nm; laser power, 0.17 mW. (c) The confocal laser image related to curve a. (d) The confocal laser image related to curve b. The collected time for both curves a and b are 15 s exposure time and 5 scans.

Transformation of D-(+)-glucose between open chain form and intramolecular hemiacetal form; Raman intensity-depth profile of 520.6 cm−1 band for a silicon wafer; simulated surface images of the substrate composed of monodisperse Ag NPs prepared at the initial pH of 10.40, the substrate composed of Au NWweaved mesoporous film prepared at the initial pH of 12.0, and the NPGL substrate; SERS spectra of 0.01 M Py acquired from the substrate composed of Au NW-weaved mesoporous film prepared at the initial pH of 12.0 and from NPGL substrate; SERS spectrum of a single yeast cell acquired after the sample has been kept for one week. This material is available free of charge via the Internet at http://pubs.acs.org.

respectively,37−39 which are related to the bioactivity of living cells.36 The peaks at 1488, 1297, and 1177 cm−1 are assigned to the deformation vibration of methyl and methylene, deformation vibration of methylene, and stretching vibration of C−O−C, respectively.37,39,40 The relative intensities of our SERS spectrum 3327



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(25) Huang, P. J.; Tay, L. L.; Tanha, J.; Ryan, S.; Chau, L. K. Chem. Eur. J. 2009, 15, 9330−9334. (26) Premasiri, W. R.; Moir, D. T.; Klempner, M. S.; Krieger, N.; Jones, G.; Ziegler, L. D. J. Phys. Chem. B 2005, 109, 312−320. (27) Zhong, Z. Y.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gedanken, A. J. Phys. Chem. B 2004, 108, 4046−4052. (28) Sylvestre, J. P.; Kabashin, A. V.; Sacher, E.; Meunier, M.; Luong, J. H. T. J. Am. Chem. Soc. 2004, 126, 7176−7177. (29) Cai, W. B.; Ren, B.; Li, X. Q.; She, C. X.; Liu, F. M.; Cai, X. W.; Tian, Z. Q. Surf. Sci. 1998, 406, 9−22. (30) Le Ru, E. C.; Blackie, E.; Meyer, M.; Etchegoin, P. G. J. Phys. Chem. C 2007, 111, 13794−13803. (31) Krauskopf, E. K.; Rice-Jackson, L. M.; Wieckowski, A. Langmuir 1990, 6, 970−073. (32) Bilmes, S. A.; Rubim, J. C.; Otto, A.; Arvia, A. J. Chem. Phys. Lett. 1989, 159, 89−96. (33) Stolberg, L.; Lipkowski, J.; Irish, D. E. J. Electroanal. Chem. 1990, 296, 171−189. (34) Jia, H. Y.; Zeng, J. B.; Song, W.; An, J.; Zhao, B. Thin Solid Films 2006, 496, 281−287. (35) Zhu, Z. Y.; Gu, R. A.; Lu, T. H. The Application of Raman Spectroscopy in Chemistry (in chinese); Northeastern University Press: Shenyang, China, 1998. (36) Li, J. F.; Huang, Y. F.; Ding, Y.; Yang, Z. L.; Li, S. B.; Zhou, X. S.; Fan, F. R.; Zhang, W.; Zhou, Z. Y.; Wu, D. Y.; Ren, B.; Wang, Z. L.; Tian, Z. Q. Nature 2010, 464, 392−395. (37) Sujith, A.; Itoh, T.; Abe, H.; Anas, A. A.; Yoshida, K.; Biju, V.; Ishikawa, M. Appl. Phys. Lett. 2008, 92, 103901. (38) Jarvis, R. M.; Brooker, A.; Goodacre, R. Anal. Chem. 2004, 76, 5198−5202. (39) Sujith, A.; Itoh, T.; Abe, H.; Yoshida, K. I.; Kiran, M. S.; Biju, V.; Ishikawa, M. Anal. Bioanal. Chem. 2009, 394, 1803−1809. (40) Osorio-Roman, I. O.; Aroca, R. F.; Astudillo, J.; Matsuhiro, B.; Vasquez, C.; Perez, J. M. Analyst 2010, 135, 1997−2001.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS We thank Jing Shen (application engineer) from HORIBA Scientific, Beijing, China, for obtaining the Raman intensitydepth profile in Figure S1, Supporting Information. We also appreciate Shukun Xu at NEU for assistance by providing live yeast cells. This work was supported by the National Natural Science Foundation of China (No. 50871028) and the Fundamental Research Funds for the Central Universities (No. N100702001 and No. N110610001). B.S. thanks the Young Scholarship Award for Doctor Candidates, Chinese Ministry of Education. G.W.Q. appreciates the Program for New Century Excellent Talents in University (No. NCET-09-0272).

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REFERENCES

(1) Smith, W. E. Chem. Soc. Rev. 2008, 37, 955−964. (2) Qian, L. H.; Das, B.; Li, Y.; Yang, Z. L. J. Mater. Chem. 2010, 20, 6891−6895. (3) Vivoni, A.; Birke, R. L.; Foucault, R.; Lombardi, J. R. J. Phys. Chem. B 2003, 107, 5547−5557. (4) Theiss, J.; Pavaskar, P.; Echternach, P. M.; Muller, R. E.; Cronin, S. B. Nano Lett. 2010, 10, 2749−2754. (5) Bechelany, M.; Brodard, P.; Elias, J.; Brioude, A.; Michler, J.; Philippe, L. Langmuir 2010, 26, 14364−14371. (6) Barhoumi, A.; Halas, N. J. J. Am. Chem. Soc. 2010, 132, 12792− 12793. (7) Lal, S.; Grady, N. K.; Kundu, J.; Levin, C. S.; Lassiter, J. B.; Halas, N. J. Chem. Soc. Rev. 2008, 37, 898−911. (8) Qian, X. M.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q. Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. M. Nat. Biotechnol. 2008, 26, 83−90. (9) Mayer, K. M.; Hafner, J. H. Chem. Rev. 2011, 111, 3828−3857. (10) Nie, S. M.; Emory, S. R. Science 1997, 275, 1102−1106. (11) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667−1670. (12) Liberman, V.; Yilmaz, C.; Bloomstein, T. M.; Somu, S.; Echegoyen, Y.; Busnaina, A.; Cann, S. G.; Krohn, K. E.; Marchant, M. F.; Rothschild, M. Adv. Mater. 2010, 22, 4298−4302. (13) Alexander, K. D.; Skinner, K.; Zhang, S. P.; Wei, H.; Lopez, R. Nano Lett. 2010, 10, 4488−4493. (14) Hong, G. S.; Li, C.; Qi, L. M. Adv. Funct. Mater. 2010, 20, 3774−3783. (15) Mulvihill, M. J.; Ling, X. Y.; Henzie, J.; Yang, P. D. J. Am. Chem. Soc. 2010, 132, 268−274. (16) Preciado-Flores, S.; Wheeler, D. A.; Tran, T. M.; Tanaka, Z.; Jiang, C. Y.; Barboza-Flores, M.; Qian, F.; Li, Y.; Chen, B.; Zhang, J. Z. Chem. Commun. 2011, 47, 4129−4131. (17) Qin, G. W.; Liu, J. C.; Balaji, T.; Xu, X. N.; Matsunaga, H.; Hakuta, Y.; Zuo, L.; Raveendran, P. J. Phys. Chem. C 2008, 112, 10352−10358. (18) Ding, Y.; Kim, Y. J.; Erlebacher, J. Adv. Mater. 2004, 16, 1897− 1900. (19) Zhang, X. Y.; Young, M. A.; Lyandres, O.; Van Duyne, R. P. J. Am. Chem. Soc. 2005, 127, 4484−4489. (20) Xu, J. J.; Zhang, L.; Gong, H.; Homola, J.; Yu, Q. M. Small 2011, 7, 371−376. (21) Guven, B.; Basaran-Akgul, N.; Temur, E.; Tamer, U.; Boyaci, I. H. Analyst 2011, 136, 740−748. (22) Wang, Y. L.; Ravindranath, S.; Irudayaraj, J. Anal. Bioanal. Chem. 2011, 399, 1271−1278. (23) Guicheteau, J.; Christesen, S.; Emge, D.; Tripathi, A. J. Raman Spectrosc. 2010, 41, 1632−1637. (24) Wang, Y. L.; Lee, K.; Irudayaraj, J. J. Phys. Chem. C 2010, 114, 16122−16128. 3328


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