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Electrochemical Synthesis and Deposition of Surface-Enhanced Raman Scattering-Active Silver Microstructures on a Screen-Printed Carbon Electrode Yang-Wei Lin, and Chung Tang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b08375 • Publication Date (Web): 15 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015

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Electrochemical Synthesis and Deposition of Surface-Enhanced Raman Scattering-Active Silver Microstructures on a Screen-Printed Carbon Electrode Yang-Wei Lin*, and Chung Tang Department of Chemistry, National Changhua University of Education, 1, Jin-De Road, Changhua City, Taiwan

E-mail: [email protected] (Y.W.L.) Tel: 011-886-4-7232105-3553

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ABSTRACT: We demonstrated a series of Ag microstructures with controlled morphologies directly deposited on a screen-printed carbon electrode by using electrochemical procedures in the presence of different electrolytes. Scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and high-resolution X-ray diffractometry were used for characterizing as-prepared Ag substrates. Thereafter, the potential of the flowerlike Ag microstructures for use in surface-enhanced Raman scattering (SERS) applications was investigated. The flower-like Ag microstructures provided a more intense SERS signal because of extremely intense local electromagnetic fields. The enhancement factor value was approximately 1.2 × 106 for 4-mercaptobenzoic acid molecules. The percentage of relative standard deviation of SERS signals was lower than 2.1%. Determining the SERS spectra of 4,4′dimercapto-azobenzene, 5,5′-dithiobis-2-nitrobenzoic acid, adenine, and single-stranded DNA (fumarylacetoacetate hydrolase gene) was straightforward. Furthermore, the thermal stability and aging behavior of the microstructures were improved. The present substrate fabrication process is facile and has excellent SERS-active properties and reproducibility, and thus, provides opportunities for quantitative analysis by using flower-like Ag microstructures. KEYWORDS: flower-like Ag microstructures; electrochemical synthesis; surface-enhanced Raman spectroscopy; 4-mercaptobenzoic acid; adenine; fumarylacetoacetate hydrolase gene

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1. INTRODUCTION Studying metal particles with well-defined nanostructures has been one of the most active research areas in recent decades.1-3 Metal nanoparticles (NPs), such as Au NPs and Ag NPs, have received considerable attention for use in fields such as chemical and biological sensing, medical diagnostics, therapeutics, and biological imaging.4-8 The sensing mechanisms of numerous applications are based on the changes in surface plasmon resonance absorbance of the metal NPs.9-10 Surface-enhanced Raman scattering (SERS) is one such application that is extremely sensitive to metal NP morphology, interparticle distance, and surrounding medium characteristics.11-12 At present, SERS is considered an extremely rapid and highly sensitive technique for studying adsorbates on surfaces, even for detecting a single molecule.13-14 Therefore, a simple method, focusing on the designation and preparation of well-ordered metal structures with specific morphologies, for fabricating SERS-active substrates is desirable.15 In particular, hierarchical metal microstructures have been attracting increasing interest because of the strong electromagnetic field enhancement for SERS measurement induced by their hierarchical characterization.15-19 Although Au is also a favorable SERS-active material because of its chemical stability, Ag always shows more favorable SERS enhancement.20-22 It is worth noting that galvanic replacement reactions or electroless deposition can also produce silver nanostructures. For example, Maboudian et al. presented an efficient, simple, and reproducible method for fabricating an Ag nanodesert rose substrate based on Ag galvanic displacement on Si.20 The authors obtained enhancement factor (EF) values of 3 × 104 for 1,2-bis(4pyridyl)ethylene, 2 × 105 for 4-mercaptopyridine, and 2 × 1010 for Rhodamine 6G compared with the evaporated flat Ag substrates. During Ag galvanic displacement, a high concentration of F− ions (0.6 M) was used. For achieving perfect galvanic displacement results, silicon chips should

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be incubated in an Ag-plating solution for 24 h. Thus, this process is unsuitable for developing environmentally benign green chemical processes and is often time consuming. Schneidewind et al. prepared Ag nanoflowers by using an enzyme-induced growth process on glass or Si substrates.21 SERS measurements of the vitamin riboflavin incubated on the Ag nanoflowers were shown as an application for quantitative analysis. Horseradish peroxidase enzyme and DNA are expensive, and chemically modifying them on Si substrates is difficult. More recently, Xia et al. proposed a hierarchical flower-like Ag microstructure through the in situ reduction of Ag+ by using a polyaniline/poly(vinyl alcohol) component.22 The rough surface of the Ag microstructure provided abundant interstitial sites, and thus, enhanced Raman signals [EF value was 105 for 4-mercaptobenzoic acid (4-MBA) molecules] were obtained. However, preparing a polyaniline/poly(vinyl alcohol) composite film requires at least 3 days. Most of the aforementioned substrate fabrication processes are restricted by a two-step method (initial complex treatment of substrates, followed by the synthesis of Ag microstructures on the substrate surface), which is often expensive and time consuming. Electrochemical method (one-step synthesis and deposition) is seen as a simple technique that provides versatility in tailoring the architecture of metals on the micro/nanoscale.23-25 However, the mechanism of the formation of the flower-like silver nanostructures by the electrochemical technique has not been fully explored.25 Furthermore, among the methods available for producing Ag flower-like structures, surfactants or templates may be required during the synthesis process. In this study, Ag microstructures with different morphologies were deposited on a screenprinted carbon electrode (SPCE) substrate through an easy one-step electrochemical process. Since SPCE is designated for mass production, the proposed process opens a useful methodology in the field of SERS-active substrates and hence the results are able extend to disposable SERS-

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active substrates. The surface morphologies of the Ag microstructures were well controlled by altering electrolyte compositions without surfacetants or templates. By using cyclic voltammetry in an AgNO3 aqueous solution containing Ag NPs, flower-like Ag microstructures were synthesized and deposited on the SPCE substrate. Because the rough surface and interlaced nanosheets of the flower-like Ag microstructures provided abundant interstitial sites, obtaining more “hot spots” with high sensitivity was easy in SERS measurement. Therefore, the EF was approximately 1.2 × 106 for 4-MBA molecules on the flower-like Ag microstructures. Moreover, the signal reproducibility, thermal stability, and aging behavior of the flower-like Ag microstructures were comprehensively investigated. For demonstrating biomolecule sensing, the label-free detection, based on sequence-selective hybridization, of single-stranded DNA (ssDNA) was performed.

2. EXPERIMENTAL 2.1. Materials. All chemicals, including 4-MBA, 4-aminothiophenol (4-ATP), 5,5′-dithiobis2-nitrobenzoic acid (DTNB), adenine, AgNO3, sodium citrate, sodium dodecyl sulfate, sodium hydrogen phosphate, disodium hydrogen phosphate, and dimethyl sulfoxide were American Chemical Society grade and obtained from Sigma–Aldrich, Co. (Milwaukee, WI, USA). Next, 0.1 M sodium hydrogen phosphate and 0.1 M disodium hydrogen phosphate were prepared in 0.1 M phosphate buffer (PB) solution (pH 7.4). Subsequently, suspensions of different-sized Ag NPs (diameter: 10.9 ± 1.8, 19.3 ± 3.7, and 56.5 ± 9.1 nm) were prepared by slightly modifying a previous technique (as shown in Figure S1).26-27 DNA sequences were purchased from Genomics (New Taipei City, Taiwan). Cy3 dye was purchased from Invitrogen (Thermo Fisher Scientific

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Inc., NY, USA). SPCEs were obtained from Zensor R&D (Taichung, Taiwan). Ultrapure water from a Milli-Q ultrapure system (Millipore, MA, USA) was used for all the experiments. 2.2. Electrochemical Synthesis of Ag Microstructures. All experiments were performed in a three-electrode system at room temperature and were controlled using a CHI 600 electrochemical workstation (CH Instruments, Austin, TX, USA). The three-electrode system comprises the SPCE as the working electrode (geometric area, 3.14 × 10−6 m2), Ag/AgCl reference electrode, and platinum wire auxiliary electrode. Before the electrochemical process, a bare SPCE was electrochemically cleaned by cycling the potential between −0.8 and 1.2 V versus Ag/AgCl in a 0.1 M PB solution (pH 7.4). The SPCE was then preoxidized by applying a potential of 0.5 V for 10 min in PB solution (pH 7.4) with stirring. Irregular-shaped Ag microstructures deposited on SPCE (substrate 1) were electrochemically synthesized by cycling the potential from −0.3 to +0.2 V versus Ag/AgCl in a 10 mM AgNO3 aqueous solution at 1.0 mVs−1 for seven scans. Under the same electrochemical conditions, flower-like Ag microstructures deposited on the SPCE (substrate 2) were fabricated in a 10 mM AgNO3 aqueous solution containing 0.1 nM Ag NPs. Next, substrate 1 and substrate 2 were roughened using an electrochemical oxidation–reduction cycle (ORC) in a 0.1 M HCl aqueous solution from −0.3 to +0.2 V versus Ag/AgCl at 5 mVs−1 for three scans (called substrates 3 and 4, respectively). All the substrates were removed from the solution and rinsed thoroughly with ultrapure water. 2.3. Characterization. A JEOL-1200EX II transmission electron microscope (TEM) (JEOL, Tokyo, Japan) was used for viewing Ag microstructures. A JSM-6510 scanning electron microscope (SEM) (JEOL) was used for measuring the size and shape of microstructures. Energy-dispersive X-ray spectroscopy (EDS, Oxford Instruments, Oxfordshire, UK) was used for confirming the compositions of microstructures. High-resolution X-ray diffraction (HRXRD)

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measurements were performed using a D8 SSS diffractometer (Bruker, Bremen, Germany) with Cu Kα radiation (λ = 0.15418 nm). 2.4. General Procedure for SERS Analysis. For SERS measurements, all substrates were incubated in 1 × 105 M 4-MBA for 30 min. The substrates were then rinsed with ultrapure water and finally dried in a vacuum dryer with dark atmosphere at room temperature for 1 h. Raman spectra were obtained using a confocal micro-Raman system (Thermo Fisher Scientific Inc.). A 532-nm laser line was used as the photoexcitation source with a laser power of 2 mW at the sample for 10 s. Raman scattering signals were collected in the backscattering geometry by using an objective lens with 10× magnitude and numerical aperture of 0.25 (laser spot size of 10 µm) and detected using a spectrometer equipped with a thermoelectrically cooled charge-coupled device detector.

3. RESULTS AND DISCUSSION 3.1 Characteristics of Various Ag Microstructures. After SPCE was performed in cyclic voltammetry experiments (+0.2 to −0.3 V vs. Ag/AgCl, 1.0 mVs−1, seven scans) in 10 mM AgNO3 solution without any additives, we observed that carbon materials on the SPCE turned silvery white, indicating the successful synthesis of metallic Ag on the SPCE surface (substrate 1). The morphology of the as-prepared Ag microstructures was identified using SEM (Figure 1a). The surface of the SPCE had many Ag microstructures with irregular morphologies. A highly magnified image of a typical Ag microstructure is also shown(Figure 1b). The EDS spectrum of the irregularly shaped Ag microstructures confirmed the presence of only Ag atoms (Figure S2a).

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Under the same conditions of cyclic voltammetry experiments, we immersed an SPCE in a 10 mM AgNO3 solution containing 0.1 nM Ag NPs and found that the SPCE turned silvery white, demonstrating metallic Ag formation on the SPCE surface (substrate 2). Figure 1c shows that the SPCE surface was also coated with many Ag microstructures, exhibiting flower-like morphologies and tending to assemble together. According to the highly magnified image of a flower-like Ag microstructure, the microstructure diameter is approximately 4.8 ± 2.1 µm (Figure 1d). These flower-like Ag microstructures, composed of different intertwined plates, produced a hierarchical structure and created abundant interstitial sites. Due to preparation limiting, these flower-like Ag microstructures were directly electrochemical synthesized on a carbon-coated Cu grid only for 3 min and characterized using TEM (Figure 2). The flower-like morphology in TEM images is consistent with that in SEM images. High-resolution TEM showed that the flower-like Ag microstructures were composed of face-centered cubic (fcc) Ag NPs because of lattice fringes at a spacing of approximately 0.25 nm.28 The EDS spectrum (Figure S2b) also confirmed that the microstructure comprised pure Ag atoms. By using the HRXRD pattern, the crystal structure and phase composition of substrates 1 and 2 were further characterized (Figure S3). Three peaks at 2θ = 38.1°, 41.2°, and 64.5° corresponded to diffractions from the (111), (200), and (220) lattice planes, respectively, of fcc Ag, revealing the presence of pure crystalline Ag microstructures. As such, the cyclic voltammetry consisting of +0.2 to −0.3 V vs Ag/AgCl, 1.0 mVs-1, and seven scans in a AgNO3 solution containing Ag NPs could be used to our subsequent electrochemical synthesis and deposition of SERS-active Ag microstructures on a glass carbon and ITO electrode. However, considering of inexpensive and disposable properties, SPCE was used for the further experiments.

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Figure 1. Low (image (a), (c), (e), and (g): scale bar = 50 µm) and high (image (b), (d), (f), and (h): scale bar = 5 µm) magnification SEM images prepared by the electrochemical procedure: (a, b) substrate 1, (c, d) substrate 2, (e, f) substrate 3, and (g, h) substrate 4.

Figure 2. (a) TEM (scale bar = 200 nm) and (b) HR-TEM (scale bar = 50 nm) images of flowerlike Ag submicrostructures fabricated using electrochemical synthesis for 3 min. The rightmost panel shows details at a high magnification (scale bar = 5 nm) with clearly observable crystallographic planes of Ag. Substrates 1 and 2 were roughened using an electrochemical ORC in a 0.1 M HCl aqueous solution from +0.2 to −0.3 V versus Ag/AgCl at 5 mVs−1 for three scans (called substrates 3 and 4). In electrochemical ORC, a 0.1 M HCl electrolyte was used because it facilitates the Ag dissolution-deposition process that yields SERS-active roughened surfaces.29 Figure S4 shows that results of the third cyclic voltammetry scan for the ORC roughening procedure. These I-E curves are quite different, indicating that the morphology of Ag microstructures would interfere with the roughening procedure. It is worthy of further study in the future. Figure 1e and g show the morphology of Ag microstructures obtained from SEM images. Both of the original

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morphologies of the Ag microstructure were clearly changed. These larger microstructures were composed of many small particles, which aggregated and formed coral-like structures (Figure 1f and h). 3.2 SERS Performance of the Developed Ag Substrates. SERS is a powerful analytical technique for the quantitative analysis of target analytes in life, environmental, and medical sciences. Substrate surface morphology is essential for this technique. As mentioned previously, the as-prepared Ag microstructures with a unique morphology and rough surface structure may prove a favorable substrate for SERS. Studies have used 4-MBA as a model molecule for evaluating SERS performance because it forms a self-assembled monolayer on the Ag surface and has been widely studied using SERS.18, 22 Figure 3 shows the SERS spectra of 4-MBA on asprepared substrates 1–4. Figure 3a shows an extremely low SERS signal for substrate 1. Substrate 2 led to peak intensities that were considerably higher than those of substrate 1 (Figure 3b). Two strong bands that dominated the SERS spectrum were at 1582 and 1076 cm−1, which were assigned to ν8a aromatic ring vibrations and ν12 aromatic ring vibrations with C−S stretching characteristics, respectively. After electrochemical ORC, compared with substrate 1, enhanced characteristic Raman spectrum peaks were clearly observed for substrate 3 (Figure 3c). Substrate 4 demonstrated SERS performance similar to that of substrate 3 (Figure 3d).

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Figure 3. SERS spectra of 10−5 M 4-MBA molecules on (a) substrate 1, (b) substrate 2, (c) substrate 3, and (d) substrate 4. By using the detectable signal, the EF values for various Ag substrates were calculated (Table 1). The EF was calculated using the following equation:

EF

       

(1)

where ISERS and Ibulk are Raman intensities obtained from SERS spectrum at 1582 cm−1 on Ag microstructures and the Raman spectrum of the bulk, respectively, and Nsurf was obtained on the assumption that the bonding density of 4-MBA in a self-assembled monolayer is 0.5 nmolcm−2.30

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The SERS-active surface area was measured using cyclic voltammetry in 5 mM Pb(NO3)2, 1 mM HClO4, and 0.1 M NaClO4 at a scan rate of 10 mV/s, assuming that a 136 µCcm−2 charge passed for the stripping of a Pb monolayer on Ag.31 Nbulk was calculated using the molecular density of 4-MBA (1.346 gcm−3) and size of the laser spot (diameter approximately 10 µm; depth approximately 10 µm), and was approximately 2.0 × 1012. The EF values were calculated according to the peak intensity at 1582 cm−1. According to the aforementioned calculation, the EF values of substrates 1, 2, 3, and 4 were 5.7 × 103, 1.2 × 106, 1.8 × 104, and 9.9 × 103, respectively. The EF value of substrate 2 is comparable to those of Ag-based SERS substrates previously reported,18, 22 indicating that substrate 2 is an effective SERS substrate.

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Table 1. Comparison of the SERS EFs of Various Ag Substrates Name Constituents Ibulk Nbulk 4-MBA 4-MBA powder spread on SPE 1246 2.0 × 1012

Substrate 1

Irregular-shaped Ag particles deposited on SPCE Substrate 2 Flower-like Ag microstructures deposited on SPCE Substrate 3 Coral-like Ag microstructures deposited on SPCE Substrate 4 Coral-like Ag microstructures deposited on SPCE a Raman intensity (peak area) is the Raman peak at 1582 cm−1 b EF defined as in Eq. (1) in the text

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ISERSa -

NSERS -

EFb -

Remarks For inherent Raman spectra of 4-MBA

23

6.5 × 106

5.7 × 103

See text for the synthesis

16360

2.1 × 107

1.2 × 106

See text for the synthesis

5647

5.0 × 108

1.8 × 104

Substrate 1 + OCR process

6780

1.1 × 109

9.9 × 103

Substrate 2 + OCR process

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Both of two effects, the surface area increased many adsorption sites and the surface morphology enhanced the electromagnetic field, are the key factors for increasing the SERS intensity. For determining the effect of electromagnetic field enhancement on surface morphology, the surface areas of various substrates were measured using cyclic voltammetry as mentioned previously. Consequently, the SERS-active surface area of substrate 2 was only 3.2times larger than that of substrate 1. However, the Raman intensity of substrate 2 at 1582 cm−1 showed a 711-fold increase compared with that of substrate 1. This result suggested that the increase in surface area was not as considerable as that in SERS intensity, and the considerable SERS enhancement effect was attributed to the electromagnetic field enhancement effect of the geometrical property of flower-like Ag microstructures. Similar results were observed for substrates 3 and 4. The surface morphology images shown in Figure 1 also support our suggestion. Therefore, the complex geometry and morphology of the Ag microstructures (substrates 2, 3, and 4) provided abundant interstitial sites, which created more “hot spots” in SERS measurement, resulting in the strong SERS effect. This effect was caused by electromagnetic enhancement mechanisms rather than chemical enhancement mechanisms. 3.3 Growth Mechanism of Flower-like Ag Microstructures. For understanding the formation of flower-like Ag microstructures on the SPCE, the morphology of metallic Ag and SERS spectra with the reaction time was investigated (Figure S5). At 1 min, no flower-like Ag microstructures on the SPCE surface were visible. At 3 min, the flower-like Ag microstructures appeared; however, they were not clearly visible because of the low surface coverage. At 5 min, a small plate-shaped structure parallel to the substrate plane held several other structures that stood on the base and interpenetrated, leading to a flower-like appearance (Figure S5a). The ongoing growth of the individual structures and the resulting increase in the surface density of

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the SPCE were clearly visible for the remaining reaction time (Figure S5b–d). As mentioned previously, the electromagnetic field enhancement in morphology, caused by the flower-like Ag microstructures, was an essential factor in enhancing the SERS intensity. The SERS signals on substrate 2 increased as the reaction time increased (Figure S5). Different-sized Ag NPs were used for fabricating Ag microstructures (Figure S6). We observed that the SERS intensity decreased when 10.9-nm Ag NPs were used. The SERS intensity was similar when 56.5-nm Ag NPs were used, likely because of the different morphology of Ag microstructures, resulting in different SERS intensities. For confirming our hypothesis, SEM was used again for observing the surface morphology of different Ag microstructures. Meatball-like Ag microstructures were observed when 10.9-nm Ag NPs were used (SEM images in Figure S6a). We know that sodium citrate acted as the reducing and capping agent for reducing Ag+ ions and then stabilizing the as-prepared spherical Ag NPs. Excess sodium citrate in Ag NP suspensions may be helpful for fabricating flower-like Ag microstructures during an electrochemical process. For proving our assumptions, Ag NP suspensions were centrifuged for obtaining supernatants and precipitates. Figure S7 shows surface morphologies and SERS spectra when the supernatants and precipitates of Ag NPs were used for fabricating Ag microstructures, respectively. The different SERS signals were caused by the different morphology and surface density of Ag microstructures on SPCE. Based on our aforementioned results, we propose a possible growth mechanism for the flowerlike Ag microstructures. The growth mechanism is based on a particle-mediated growth mechanism,17,

19

which can be divided into three stages. In the first stage, Ag+ ions are

electroreduced into Ag atoms by cyclic voltammetry (scanned to cathodic vertex potential) and a chemical reductant (sodium citrate in the Ag NP suspension). As the reduction progresses, the

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concentration of Ag atoms on the SPCE surface gradually increases. In the second stage, as the concentration of Ag atoms exceeds the supersaturation point of nucleation, the atoms aggregate to form a nucleus. Thus, Ag NPs are fabricated on the SPCE surface. Simultaneously, the concentration of Ag atoms decreases because the concentration of Ag NPs increases. The formation of Ag NPs stops when the concentration of Ag atoms decreases to below the supersaturation point of nucleation. In the reaction system, sodium citrate performs as a chemical reductant as well as a capping agent. Ag NP suspension can prevent the oxidation of Ag NPs which are already deposited on the SPCE surface. Furthermore, it also can promote the aggregation of Ag NPs on the SPCE surface during cyclic voltammetry scanned to anodic vertex potential. In the third stage, the Ag NPs aggregate to form flower-like microstructures through particle-mediated growth.17, 19 The remaining Ag+ ions are electroreduced and electrodeposited on the particles, thus inducing the overgrowth of the flower-like Ag microstructures when cyclic voltammetry is scanned again to cathodic vertex potential. 3.4 Optimization of the SERS Performance of Flower-like Ag Microstructures. Additional assay parameters were evaluated for further optimizing the SERS performance for substrate 2. The concentration effects of Ag NPs used for fabricating substrates were tested at 0.05, 0.1, and 0.2 nM (Figure S8); the SERS intensity of 4-MBA increased with Ag NP concentrations ranging from 0.05 to 0.1 nM, above which the intensity slightly decreased. This result was likely because of high Ag NP concentrations that favor the formation of large individual Ag structures on the SPCE substrate surface. However, a large individual structure cannot easily adsorb on the SPCE surface, resulting in decreasing SERS intensity. For confirming this hypothesis, SEM was used for calculating the surface density of various substrates prepared at different Ag NP concentrations. The substrate prepared using 0.1 nM Ag

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NPs had the highest surface density of Ag microstructures on the SPCE. Therefore, 0.1 nM Ag NPs were selected as the optimal concentration for subsequent investigations. Different Ag+ ion concentrations ranging from 2 to 50 mM (Figure S9) were also tested; the SERS intensity of 4MBA increased with Ag+ ion concentrations up to 10 mM, above which the intensity decreased. This result was likely because of the size and morphology changes in Ag microstructures at different concentrations. For confirming this hypothesis, SEM was used again for viewing the size and morphology of different Ag microstructures. Small Ag microstructures were obtained at high Ag+ ion concentrations. At 50 mM Ag+ ions, the morphology of Ag microstructures clearly changed. Therefore, 10 mM was chosen as the optimal Ag+ ion concentration. For testing the effect of the pre-oxidation potential, different potentials ranging from 0.25 to 10 V were tested. The maximum SERS intensity of 4-MBA was obtained when the oxidation potential was 0.5 V (Figure S10a). Subjecting the SPCE at high pre-oxidation potentials produces more hydroxyl groups on the SPCE surface, favoring Ag+ ion adsorption.32 In addition, the hydroxyl groups also act as electron donors and aid in the formation of large Ag microstructures. However, large individual structures cannot easily adsorb on the SPCE surface, resulting in decreasing Raman intensity. Therefore, the pre-oxidation potential of 0.5 V was selected for subsequent experiments. In addition, the influences of scan ranges, rates, and numbers on the preparation process were tested. The maximum SERS intensity was demonstrated when the SPCE was prepared at the cathodic and anodic vertex potentials of −0.3 and +0.2 V versus Ag/AgCl, respectively (Figure S10b). The reduction reaction on the electrode at the cathodic vertex of −0.3 V was considerable.33 The formation of Ag microstructures did not occur at a reduced cathodic vertex potential. A high scan rate of the electrochemical process resulted in small Ag microstructures, and, consequently the surface density on SPCE decreased,

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thus decreasing the SERS intensity (Figure S10c). A high scan number increases the surface density of Ag microstructures on the SPCE surface, thus increasing the SERS intensity (Figure S10d). However, in this study, substrate fabrication required more than 4 hrs. Therefore, the optimal scan rate and number were 1.0 mVs−1 and seven, respectively. Figure 4 shows the SERS spectra when different concentrations of 4-MBA solutions were tested at the optimal conditions for substrate 2. At 0.1 µM, 4-MBA did not show any characteristic peaks, and thus, the 1.0 µM concentration was considered the limit of quantity (both the characteristic vibrations at 1582 and 1076 cm−1 could be obtained). A linear relationship was obtained from the plot of Raman intensity at 1582 cm−1 as a function of the concentration of 4-MBA molecules ranging from 1.0 to 10 µM (R2 = 0.99). Figure S11a shows the Raman mapping images deomstrating the SERS reproducibility. In addition, the SERS spectra of 4-MBA for 10 substrate 2 samples are shown in Figure S11b. The percentage of relative standard deviation (RSD%) of SERS signals was less than 2.1%, indicating the high signal reproducibility of the prepared substrate 2. Some intensity fluctuations were caused by variations in molecule adsorption. Nevertheless, the SERS signals were relatively stable, demonstrating

a

favorable

SERS-active

substrate

fabricated

using

the

controllable

electrochemical technique.

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Figure 4. SERS spectra of different concentrations of 4-MBA adsorbed on substrate 2. Inset: The plot of Raman intensity at 1582 cm−1 as a function of the concentration of 4-MBA molecules. 3.5 Thermal Stability and Aging Behavior of Flower-like Ag Microstructures. Because a long acquisition time (> 2 hrs) was used for obtaining favorable spectroscopic results, the destructive thermal effect on the SERS capability of the prepared substrate 2 was evaluated using long-term laser irradiation. At 150 °C, thermogravimetric analysis showed a 12% weight loss of 4-MBA molecules; at 100 °C, the weight loss was only 5%. This finding indicated the slight decomposition of 4-MBA at high temperatures. Figure 5(e) shows the SERS spectra of 4-MBA adsorbed on substrate 2 at 25, 50, 100, and 150 °C. The SERS signal was greatly decreased at 50 °C from 25 °C. As shown in the literature, substrate-temperature dependences of SERS below and above room temperature were widely investigated through theoretical and experimental

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studies.34-36 We propose a possible mechanism in which high temperature induced a small but significant particle diffusion in closely spaced Ag microstructures. The result of the shifting changed the average interparticle distance, which subsequently decreases the electromagnetic coupling between the Ag microstructures, and in turn causes a decrease in the SERS intensity. The operating temperature for the prepared substrate 2 could be increased to 100 °C. When the temperature was increased to 150 °C, the characteristic Raman peaks of 4-MBA could not be defined, as shown by the green spectrum in Figure 5(e). The SEM images in Figure 5(a)–(d) also show that the decrease in the SERS capability at 150 °C was due to the destruction of substrate 2. The originally flower-like Ag microstructures become smoother, and some thin films were observed due to annealing effects.33, 37 These results indicate that the loss in SERS effects for the flower-like Ag microstructures can be attributed to the decrease of the electromagnetic field enhancement effect, as revealed from the SEM images.

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Figure 5. SEM images (scale bar = 5 µm) of 4-MBA on substrate 2 at (a) 25 °C, (b) 50 °C, (c) 100 °C, and (d) 150 °C and (e) SERS spectra of 4-MBA on substrate 2 at different temperatures. For testing the aging behavior of substrate 2, it was placed in an atmosphere of 50% relative humidity and 20% O2 at 25 °C for 54 days. The normalized Raman intensity was calculated from

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the ratio of the peak intensity of 4-MBA at 1582 cm−1 adsorbed on the prepared substrate 2 at the first day to that of 4-MBA adsorbed on an as-prepared substrate 2 at measuring time. The normalized intensities of 4-MBA adsorbed on substrate 2 were maintained at 80% for 54 days (Figure 6). This finding shows that substrate 2 can provide favorable SERS performance with regard to stability and durability. We also demonstrated increases in the normalized Raman intensity during the first 5 days for substrate 2. This phenomenon is also reported by Liu’s group and presently under investigation in our lab.38-39

Figure 6. Variation in the normalized Raman intensity of 4-MBA adsorbed on substrate 2 in 50% RH and 20% O2 at 25 °C for 54 days.

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3.6 Applications of Flower-like Ag Microstructures. Figure 7a shows the SERS spectrum of DTNB, which has characteristic Raman peaks at 1064, 1153, 1336, and 1557 cm−1.40 The strong peak at 1336 cm−1 was assigned as the symmetric stretch of the nitro group and that at 1557 cm−1 was assigned to an aromatic ring mode. The photochemical conversion of 4-ATP to 4,4′-dimercaptoazobenzene (DMAB) occurred on the substrate 2 surface.41 Figure 7b shows the SERS spectrum of DMAB, whose Raman peaks at 1074 and 1581 cm−1 were attributed to the ring breathing coupled with C–S and C–C stretching vibrations, respectively. The Raman peaks at 1141 and 1187 cm−1 were attributed to C–N stretching and C−H bent vibrations, respectively. Furthermore, the Raman peaks at 1390 and 1435 cm−1 were assigned to N–N and C–C stretching vibrations, respectively. The strong SERS signals on substrate 2 were caused by “hot spots” that were generated at the junction between Ag surfaces after irradiation with light. This major SERS effect was ascribed to the electromagnetic enhancement and was relative to the Ag microstructure geometry. This strongly enhanced Raman effect showed that substrate 2 was capable of molecular sensing with high sensitivity.

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Figure 7. SERS spectra of (a) DTNB, (b) 4-ATP, (c) adenine, and (d) Cy3 from dsDNA adsorbed on substrate 2. For demonstrating the biomolecule application of substrate 2, it was used for detecting adenine and ssDNA. Ten microliters of a 1 µM adenine solution was dropped and dried on substrate 2. Figure 7c shows the SERS spectrum of adenine, which clearly showed Raman peaks at 731, 1323, and 1395 cm−1, indicating that substrate 2 can be used for detecting biomolecules at low concentrations.15 Next, ssDNA was detected using substrate 2. A thiol-terminated DNA strand (5′-CCAGATACTCACCGG-SH-3′)

was

used

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a

probe,

which

recognizes

the

fumarylacetoacetate hydrolase gene.42 Mutation of this gene is associated with a human genetic

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disease, hereditary tyrosinemia type 1. Through strong Ag–S covalent bonding, this probe was immobilized overnight on the substrate 2 surface at room temperature. After incubation, excess probe was washed with PB solution containing 0.2% sodium dodecyl sulfate for 3 min. The modified substrate 2 was incubated with 1 µM target ssDNA (5′-CCGGTGAGTATCTGG-3′) in the PB solution containing 10 µM Cy3 dye as for the Raman reporter. After washing with PB solution, the SERS spectrum of the modified substrate 2 was measured. A strong SERS spectrum of Cy3 dye was observed for complementary target ssDNA, with a limit of quantity of 10 nM. A linear relationship was also obtained from the plot of Raman intensity (1467 cm−1) as a function of the concentration of target DNA strand over the range of 10 nM–10 µM (R2 = 0.955). For noncomplementary ssDNA, no double-strand DNA formation was observed. Thus, Cy3 molecules could not intercalate with ssDNA, and a featureless spectrum was thus demonstrated. Therefore, after additional optimizations, substrate 2 has great potential in DNA detection techniques.

4. CONCLUSIONS We demonstrated an easy one-step electrochemical approach for synthesizing flower-like Ag microstructures deposited on SPCE substrates and the application of these microstructures in SERS. Abundant interstitial sites are formed because of the rough surface and interlaced nanosheets of the flower-like Ag microstructures exhibiting high sensitivity in the SERS measurement. Compared with other approaches, our proposed approach possesses attractive features and advantages (Table 2), such as being (1) inexpensive, and environmentally friendly (expensive enzymes, organic solvents, templates, surfacetants, and complex substrate preparation methods are not required); (2) time saving (fabrication time requires only 2 hrs); (3) reproducible

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(RSD% values for 10 Ag substrates were less than 2.1%); (4) durable (flower-like Ag microstructures have reduced losses of SERS effects at 100 °C and can withstand aging for 54 days); and (5) practical (high-quality SERS spectra were obtained for various molecules, such as 4-MBA, DMAB, DTNB, adenine, and ssDNA). Because finding advanced SERS-active substrates continues to be an active research area, our proposed simple method could help in designing novel SERS-active substrates. In addition, the flower-like Ag microstructures deposited on the SPCE can be also used with an electrochemical analytical system for environmental and biological sensing applications. For exmaple, under electrochemical condition, the analyte is first reduced (or oxidized) and then deposited on the Ag microstructure-modified SPCE, usually from a stirred solution. After a measured period, the electrolysis is discontinued, the stirring is stopped, and the deposited analyte is determined by Raman spectroscopy. As a result of the pre-concentration step, the integrated systems yield the lowest detection limits.

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Table 2. Comparison of the Preparation Process, EF Values, Durability, and Applications for the Flower-Like Ag Microstructures Substrate Preparation process and time EF values Durability Practicality Ref. a 20 10 Ag nanodesert rose on Si Galvanic displacement process NPE, 4-MPy, 2.0 × 10 for R6G (one-step method )/24 h R6G a a 21 Nanoflower-like Ag NPs Enzyme-induced growth process Riboflavin on glass substrates (two-step method)/4 h 22 Flower-like Ag Chemical reduction on -a 4-MBA 1.9 × 105 for 4microstructures on a glass PANI/PVA composite film (two- MBA plane step method)/3 days Flower-like Ag Electrochemical reduction on 100 °C/54 4-MBA, DMAB, This 1.2 × 106 for 4microstructures on SPCE SPCE/2 hrs days DTNB, adenine, study MBA FAH gene a not provided

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AUTHOR INFORMATION ACKNOWLEDGMENT This study was supported by the Ministry of Science and Technology under contract (MOST 103-2113-M-018-001-MY2). We thank Wallace Academic Editing for the English language editing.

Supporting Information Available: Figure S1. TEM images of Ag NPs with different sizes: (a) 10.9 ± 1.8 nm, (b) 19.3 ± 3.7 nm, and (c) 56.5 ± 9.1 nm. Scale bar = 5 nm for image (a)–(b) and scale bar = 10 nm for image (c). Figure S2. EDS spectra for (a) substrate 1 and (b) substrate 2. Figure S3. High-resolution X-ray diffraction spectra of (a) substrate 1 and (b) substrate 2. Figure S4. Cyclic voltammograms of the third scan for ORC roughening procedure of substrate 1 (black) and substrate 2 (red). Figure S5. SERS spectra of 10−5 M 4-MBA molecules on substrate 2 prepared with various electrodeposition times. Representative SEM images of substrate 2 with various electrodeposition times: (a) 5, (b) 15, (c) 30, and (d) 45 min. Scale bar = 2 µm for image (a) and scale bar = 5 µm for image (b)–(d). Figure S6. SERS spectra of 10−5 M 4-MBA molecules on Ag microstructures fabricated in a 10 mM AgNO3 aqueous solution containing 0.1 nM (a) 10.9-nm and (b) 56.5-nm Ag NPs. The rightmost images show low (scale bar = 50 µm) and high (scale bar = 10 µm) magnification SEM images for (a) and (b), respectively. Figure S7. SERS spectra of 10−5 M 4-MBA molecules on Ag microstructures prepared in 10 mM AgNO3 aqueous solution containing the (a) precipitates and (b) supernatants of 0.1 nM Ag NP (diameter: 19.3 ± 3.7 nm) suspensions. The rightmost images show low (scale bar = 50 µm) and high (scale bar = 5 µm) magnification SEM images for (a) and (b), respectively. Figures S8. SEM images

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and Raman intensities at 1582 cm−1 of 10−5 M 4-MBA molecules on substrate 2 prepared with various Ag NP concentrations: (a) 0.05, (b) 0.1, and (c) 0.2 nM. Scale bar = 50 µm. Figure S9. SEM images and Raman intensities at 1582 cm−1 of 10−5 M 4-MBA molecules on substrate 2 prepared with various Ag+ ion concentrations: (a) 5, (b) 10, (c) 25, and (d) 50 mM. Scale bar = 50 µm.

Figure S10. Raman intensities at 1582 cm−1 of 10−5 M 4-MBA adsorbed on Ag

microstructures with different (a) pre-oxidation potentials, (b) scan ranges, (c) scan rates, and (d) scan numbers. Figure S11. Reproducibility test for substrate 2. (a) Raman mapping over the substrate 2, and (b) ten substrates were randomly selected for measuring the SERS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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41. Zhao, L. B.; Huang, Y. F.; Wu, D. Y.; Ren, B. Surface-Enhanced Raman Spectroscopy and Plasmon-Assisted Photocatalysis of p-Aminothiophenol. Acta Chim. Sinica 2014, 72, 11251138. 42. Liu, C. W.; Lin, Y. W.; Huang, C. C.; Chang, H. T. Fluorescence Detection of SingleNucleotide Polymorphisms Using a Thymidine-Based Molecular Beacon. Biosens. Bioelectron. 2009, 24, 2541-2546.

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TOC

SERS detection of FAH gene using flower-like Ag microstructures

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Figure 1. Low (image (a), (c), (e), and (g): scale bar = 50 µm) and high (image (b), (d), (f), and (h): scale bar = 5 µm) magnification SEM images prepared by the electrochemical procedure: (a, b) substrate 1, (c, d) substrate 2, (e, f) substrate 3, and (g, h) substrate 4. 375x507mm (96 x 96 DPI)

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Figure 2. (a) TEM (scale bar = 200 nm) and (b) HR-TEM (scale bar = 50 nm) images of flower-like Ag submicrostructures fabricated using electrochemical synthesis for 3 min. The rightmost panel shows details at a high magnification (scale bar = 5 nm) with clearly observable crystallographic planes of Ag. 457x150mm (96 x 96 DPI)

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Figure 3. SERS spectra of 10−5 M 4-MBA molecules on (a) substrate 1, (b) substrate 2, (c) substrate 3, and (d) substrate 4. 415x307mm (96 x 96 DPI)

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Figure 4. SERS spectra of different concentrations of 4-MBA adsorbed on substrate 2. Inset: The plot of Raman intensity at 1582 cm−1 as a function of the concentration of 4-MBA molecules. 463x314mm (96 x 96 DPI)

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Figure 5. SEM images (scale bar = 5 µm) of 4-MBA on substrate 2 at (a) 25 °C, (b) 50 °C, (c) 100 °C, and (d) 150 °C and (e) SERS spectra of 4-MBA on substrate 2 at different temperatures. 448x509mm (96 x 96 DPI)

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The Journal of Physical Chemistry

Figure 6. Variation in the normalized Raman intensity of 4-MBA adsorbed on substrate 2 in 50% RH and 20% O2 at 25 °C for 54 days. 448x314mm (96 x 96 DPI)

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Figure 7. SERS spectra of (a) DTNB, (b) 4-ATP, (c) adenine, and (d) Cy3 from dsDNA adsorbed on substrate 2. 415x321mm (96 x 96 DPI)

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The Journal of Physical Chemistry

TOC. SERS detection of FAH gene using flower-like Ag microstructures 431x274mm (96 x 96 DPI)

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