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nanostructure clusters on Cu substrates for in-situ pre-concentration and surface-enhanced Raman scattering
Accepted Manuscript Title: Inkjet-printed Ag Micro-/Nanostructure Clusters on Cu Substrates for In-situ Pre-concentration and Surface-Enhanced Raman Scattering Author: Qitao Zhou Ashish Kumar Thokchom Dong-Joo Kim Taesung Kim PII: DOI: Reference:
S0925-4005(16)31932-3 http://dx.doi.org/doi:10.1016/j.snb.2016.11.134 SNB 21344
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Sensors and Actuators B
Received date: Revised date: Accepted date:
16-9-2016 17-11-2016 24-11-2016
Please cite this article as: Qitao Zhou, Ashish Kumar Thokchom, Dong-Joo Kim, Taesung Kim, Inkjet-printed Ag Micro-/Nanostructure Clusters on Cu Substrates for Insitu Pre-concentration and Surface-Enhanced Raman Scattering, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.11.134 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inkjet-printed Ag Micro-/Nanostructure Clusters on Cu Substrates for In-situ Pre-concentration and Surface-Enhanced Raman Scattering Qitao Zhou, Ashish Kumar Thokchom, Dong-Joo Kim and Taesung Kim*
Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulsan 44919, Republic of Korea.
*Corresponding author E-mail: [email protected] Department of Mechanical Engineering Ulsan National Institute of Science and Technology (UNIST) 50 UNIST-gil, Ulsan 44919, Republic of Korea.
1
Graphical abstract Surface-enhanced Raman scattering is a promising technology for biosensing but the challenge of creating practical substrates remains. This study presents a novel inkjet printing technique for fabricating (SERS) substrates that offer both in-situ fabrication of the noble metal nanoarrays, and thus high density SERS “hot spots” but also pre-concentration of the target analyte.
Highlight:
A novel inkjet printing technique for fabricating SERS substrates is presented. In-situ fabrication of noble metal nanoarrays for high density SERS hot spots and wettability gradients for pre-concentration of target analytes. Highly sensitive SERS detection of antibiotics at very low concentrations below 100 pM.
Abstract Effective surface-enhanced Raman scattering (SERS) detection requires substrates that are typically fabricated
using
expensive,
low-throughput
and
time-consuming
micro-
/nanofabrication processes such as photolithography, electron-beam lithography and templateassisted methods. Here, a novel micro-/nanofabrication technique for fabricating SERS substrates with hydrophobicity gradients is demonstrated. An inkjet printer enables injecting an AgNO3 solution onto a thiol-functionalized superhydrophobic Cu surface, upon which Ag micro/nanostructures are generated via replacement reactions in the droplet-injected areas. When a mixed solution of target analytes and Au-nanoparticles (Au-NPs) are placed on this 2
substrate, the contact area decreases over time due to the evaporation of the solution and the hydrophobicity of the substrate. As a result, the analyte molecules and Au-NPs are delivered to the Ag micro-/nanostructure clusters, upon which the analyte and Au-NPs are simply and easily concentrated in situ. With the cooperation of Ag nanoplates and Au-NPs, two antibiotics at very low concentrations (e.g., 100 pM 6-aminopenicillanic acid and 50 pM penicillin G sodium) were successfully detected, confirming the higher SERS activity than that of Ag-nanoplate-assembled nanotube arrays or an Ag-NPs decorated graphene electrophoretic pre-concentration device. Hence, this rapid design-to-prototype method for substrates with adjustable wetting properties can be very useful for a SERS platform to detect various target analytes in biosamples.
Keywords: inkjet printing, pre-concentration, wettability, surface-enhanced Raman scattering (SERS), analyte detection
1. Introduction Surface-enhanced Raman scattering (SERS) is of particular interest for detecting and monitoring hazardous and/or harmful molecules, because it can rapidly detect analytes while simultaneously providing their mass fingerprint spectra. Widely used mass-based technologies such as gas chromatography with mass spectrometry (GC–MS),[1] or mass spectrometry (LC–MS)[2] can elucidate the chemical structures and mass fingerprints of molecules, but they usually require complex extractions and purification procedures, which are both time- and sample-consuming.[3] On the other hand, more rapid detection methods such as enzyme-linked immunosorbent assays (ELISA)[4] and fluorescent sensors[5] are 3
usually based on specific biochemical or physicochemical interactions and cannot provide mass fingerprints. For effective SERS detection, plasmonic metal nanostructures are used to amplify the sensing signal.[6-8] Thus, various noble metal nanostructures have been proposed. For example, Au and Ag colloidal nanoparticles have been used as platforms to achieve high SERS-activity from “hot spots” (i.e., sub-10-nm-gaps between the noble metal nanoparticles) in randomly dispersed colloidal systems;[9-13] however, the reproducibility of SERS signals from different locations is poor, thus restricting SERS-based practical applications. Regularly patterned arrays of noble metal nanoparticles[14] and, nanorods[15] and Ag semishells[16] were fabricated with the assistance of templates such as porous anodic aluminum oxide (AAO) or colloidal polystyrene (PS) spheres to achieve good SERS signal reproducibility with high sensitivity, as high density “hot spots” could be homogeneously distributed on the whole patterned substrate. However, these template-assisted methods usually have several impractical steps and are time consuming. Developing a template-free fabrication method for noble metal micro/nanostructure arrays has considerable practical significance. Recently, inkjet printing has been developed into an effective method for fabricating micro-/nanostructure arrays as this type of printing can simply drop the ink solution in a fixed volume onto a set position.[17-20] Based on these advantages, Ag or Au nanoparticles have been printed on cellulose paper or silicon wafers for use as SERS substrates.[21, 22] However, in these cases, the nanostructures in the inks, such as colloidal Ag nanoparticles, needed to be synthesized in advance. Therefore, inkjet printing redistributed the nanostructures rather than fabricating them, which does not fully exploit the advantages of inkjet printing, such as its simplicity and efficiency. Thus, developing an inkjet-printing-based method that offers in-suit fabrication of noble metal micro-/nanostructure would be a significant step forward. On the other hand, to enable sensitive SERS detection, as many analyte molecules as possible should be in the vicinity of the plasmonic nanostructure surface so that the SERS-active molecules 4
are exposed to the strong electromagnetic field generated by the plasmonic nanostructures.[2326]
For instance, mono-6-thio-β-cyclodextrin has been functionalized onto a SERS substrate to
capture analyte molecules such as polychlorinated biphenyls and methyl parathion. [27,
28]
However, the trapping effect of cyclodextrin depends on the design of the cyclodextrin cavity diameter,[29] which is complicated and time-consuming. Alternatively, macromolecules such as proteins, antibodies and DNA immobilized on SERS substrates have been employed to capture the antigen or analytes,[30-32] but such samples are difficult to prepare and preserve. To solve these problems, SERS substrates with a hydrophobicity gradient have been designed and prepared. This hydrophobicity gradient is intended to draw all the molecules in a sample into the detection region of the SERS substrate. For example, electron-beam lithography (EBL) and focused ion beam (FIB) milling were used to fabricate SERS-active elements and structures for superhydrophobicity. During the detection, analyte molecules in the droplet were extracted to the SERS-active Ag-decorated nanocone in the center, thus realizing SERS detection at ultra-low concentrations.[33] To make the fabrication process simpler and less expensive, optical lithography have has been used rather than EBL and FIB to fabricate a superhydrophobic bull's-eye substrate. After a water droplet containing a gold nanoparticle colloid was pipetted onto the superhydrophobic bull's-eye substrate, the gold nanoparticles aggregated at the center to act as the SERS active region.[34] Nevertheless, this approach is still not sufficiently simple or inexpensive for a ready-to-use SERS substrate. Thus, developing a SERS substrate with a hydrophobicity gradient using a template-free fabrication method remains a challenge. In this work, we describe a novel, simple and convenient method to fabricate metal micro/nanostructure clusters using an inkjet printer. We optimize the concentration of a silver nitrate (AgNO3) solution as an ink, and we then characterize the Ag micro-/nanofabrication process in which Ag nanoplate clusters (Ag-NPCs) are created via the substitution reaction between Cu and AgNO3.[35] Furthermore, we characterize the surface wettability effect by 5
adjusting the spacing distance between the Ag-NPCs, thus functionalizing the Cu surface to endow it with a hydrophobicity gradient. Recently, the development of reliable analytical techniques to detect antibiotic residues in the environment has garnered considerable interest, as the needless overuse of antibiotics can lead to drug resistance, toxicity, and allergic reactions.[36] Therefore, we demonstrate a novel SERS detection platform for antibiotics by exposing the engineered hydrophobicity gradient to droplets of target analytes and Au nanoparticles (Au-NPs), which are then pre-concentrated to enhance the SERS activity. Lastly, we compare the performance of our platform with previous SERS-based detection approaches. 2. Results and Discussion 2.1. Fabrication of Ag-NPCs on thiol-decorated Cu substrates Figure 1 shows the substrate fabrication steps. Because the hydrophobicity gradient plays an important part in pre-concentrating the target analytes, we modified the surface wettability of the Cu surface using a thiol coating, and we regulated the printing conditions of AgNO3. As shown in Figure 1a, the contact angle (CA) of water on a bare Cu substrate was 97°, indicating the relatively weak hydrophobicity of the bare Cu substrate. To achieve a superhydrophobic surface, we functionalized the substrate in n-octadecanethiol solution in N,N-dimethylformamide (DMF), as described below in the Experimental section. The low surface energy of the surface bonded n-octadecanethiol significantly increased the hydrophobicity of the bare Cu substrate. The morphology of the Ag-NPCs synthesized via inkjet printing was measured using scanning electron microscopy (SEM). As shown in Figure 1b, thiol modification increased the CA to 155°. Then, a AgNO3 solution was injected using the inkjet printer onto the superhydrophobic Cu surface. As Cu has higher reactivity, the Ag ions were reduced to Ag nanoplates, and the Ag nanoplates grew from the Cu surface, as shown in Figure 1c. In turn, regular Ag-NPCs composed of interlaced Ag nanoplates formed on the Cu substrate surface. The formation of metallic Ag was confirmed by energy dispersive X-ray spectroscopy (EDS) (Figure S1 in the Supporting Information). 6
2.2. Effect of print spacing on the wettability of Ag-NPC arrays After printing on the designated areas of the thiol-decorated Cu substrate, the hydrophobicity of these areas will be reduced. The inset in Figure 1c shows that the CA changed from 155° to 151°due to these Ag-NPC arrays. In order to demonstrate the role of Ag-NPC arrays in regulating the wettability of the surface, the bare Cu substrate have was printed with Ag micro-/nanostructure arrays at various distances between the centers of neighboring droplets. The CA could be adjusted from 67° to 53° as the distances between the arrays were decreased from 120 μm to 60 μm and 40 μm (Figure S2). As the distance between the hydrophilic Ag clusters decreased, the amount of the Cu surface covered by the clusters (i.e., the surface density of the clusters increased), thus decreasing the CA. Therefore, the AgNPCs can increase the hydrophilicity of the surface. Similarly, a region with lower hydrophobicity can be obtained in the droplet printed areas on the superhydrophobic Cu surface. To achieve a steeper hydrophobicity gradient and to ensure that the AgNO3 droplets would not interact with each other, 40 μm was set as the printing distance in the subsequent experiments. Interestingly, if the thiol-treatment step was implemented after inkjet printing, the hydrophobicity of the Cu substrate could be further improved (Figure S3). The obvious increase in the CA (from 151° to 168°) was due to the increasing amount of surface area with a low surface energy. This increase occurred because when the Cu substrate decorated with Ag-NPCs was immersed into the thiol solution, the thiol molecules labeled both the Cu and Ag surfaces. 2.3. Effect of AgNO3 concentration on SERS activity As shown in Figure 2, the size and SERS activity of the Ag nanoplates can be tuned by adjusting the concentration of AgNO3. With the decrease in AgNO3 concentration from 0.5 M to 0.1 M, the thickness of the Ag nanoplates decreased, but the density of the Ag nanoplates increased, leading to smaller gaps and the number increase of nano-gaps (less than 10 nm, i.e., the desired so-called “hot spots”) increased between nearest neighboring Ag nanoplates. 7
Therefore, more “hot spots” were achieved, which improved the SERS activity (Figure 2a-e). However, with a further decrease in the AgNO3 concentration, the Ag nanoplates became smaller and sparser, and therefore, the “hot spots” in the small gaps decreased between neighboring Ag nanoplates, leading to a decrease in the SERS activity, as shown in the upper two spectra in Figure 2f. As shown in Figure 2g, the Ag-NPC-decorated Cu substrate prepared with 0.1 M AgNO3 showed the strongest SERS activity. Thus, it was used as the SERS substrate in the subsequent experiments. As shown in Figure S4, SERS mapping images were obtained from the five samples prepared by different AgNO3 concentrations for the objective comparison of SERS activity. It was confirmed that the signal intensities changing with the AgNO3 concentrations show the similar tendency as Figure 2g. 2.4. Pre-concentrating and fixing the analyte via evaporation Figure 3a shows the drying process of a water droplet placed on the functional Cu substrate over time. As water evaporates from the drop, its volume shrinks. However, its shape remains, and it is fixed at the same center of the substrate. During most of this drying process, the CA of the droplet remains almost constant. However, further drying eventually results in sharp reductions in the CA. When the water droplet is replaced by an analyte solution, the molecules in the solution can be concentrated into a small area, as illustrated in Figure 3b. This is beneficial to SERS detection because the higher the analyte concentration, the more sensitive the SERS detection. Not only can the hydrophobicity gradient preconcentrate the analyte, but it can also help fix the analyte droplet on the surface. Notably, the Ag-NPC-decorated Cu surface could immobilize the solution droplet at a fixed place (refer to Supplementary Movie S1). Specifically, when the substrate is tilted at an increasing angle, the solution droplet stays in the same place on the Ag-NPC-printed substrate; meanwhile, on the unprinted thiol-decorated superhydrophobic Cu substrate, the droplet slides down. This result shows the difference between the wettability of the thiol-decorated Cu surface and the Agnanoplate-cluster-decorated Cu substrate, displaying its ability to capture droplets. In addition, 8
many small droplets containing analyte molecules can be fixed on the substrate without interfering with each other, implying that different concentrations of different analytes can be detected on a single SERS substrate. As shown in Figure 3c, 5 μL droplets of different dye solutions at various concentrations were dripped one by one onto the regular 2 mm × 2 mm printed patterns on a 2 cm × 2 cm Cu substrate, and the droplets fixed onto the printed patterns very well. 2.5. Decorating the Ag-NPCs with Au-NPs As shown in Figure 4, we tested the SERS ability of the Ag-NPCs by incorporating the Au-NP solution. After evaporation, a visible ring pattern formed caused by a coffee-ring effect (Figure 4a(i)).[22, 37] Outside the ring, there are almost no Au-NPs (Figure 4a(ii)). Due to the difference between the wettability of the thiol-decorated Cu surface and that of the AgNPCs, the Au-NPs preferentially deposited on the Ag nanoplates. The SEM image taken from the area in the coffee ring (Figure 4a(iv)) shows that some Au-NP clusters were randomly distributed around the Ag-NPC. Accordingly, a large number of Au-NPs deposited on the Ag nanoplates due to their hydrophilicity. As schematically shown in Figure 4b, to compare the SERS ability of Ag-NPCs decorated by Au-NPs (Ag-NPC/Au-NPs) and Au-NPs alone on the Cu surface, six spots were randomly chosen on the substrate to measure. Comparing the average signal intensities at 612 cm-1 in the two typical SERS spectra (Figure 4c-d), the signal taken from Ag-NPCs/Au-NPs is almost 6 times more intense than that of the Au-NPs alone on the Cu surface. This higher intensity is because not only can the Ag nanoplates supply “hot spots”, but also because there are many “hot spots” between the Au-NPs. In addition, rather than dropping the analyte solution on the SERS substrate, we measured the SERS signals by immersing the substrate in the analyte solution and then drying it for comparison (see Figure S5). It turned out that the signal intensities taken from the dropped sample are at least 3 times higher than those taken from the immersed sample, verifying that the pre-concentration of the analyte driven by a hydrophobicity gradient is much stronger than the self-adsorption of the 9
analyte to the SERS substrate. Furthermore, the plasmonic coupling between the Ag nanoplates and the Au-NPs can also further improve the SERS ability. Figure S6 shows a SERS mapping image taken from the Ag-NPCs/Au-NPs on a substrate in which the central part with the cooperation of Ag-NPCs and Au-NPs exhibits much stronger and uniform SERS signals. In contrast, lower and nonuniform signals are taken from the rest part as there are only some randomly distributed Au-NPs. 2.6. SERS performance with and without Au-NPs We compared the SERS performance of an Ag-NPC-decorated Cu substrate with that of the Ag-NPC-decorated Cu substrate with Au-NPs using a well-known molecule (e.g., rhodamine 6G [R6G]), as shown in Figure S7. Although the Ag-NPC-decorated Cu substrate can successfully detected R6G down to 100 pM, we aimed to enhance the sensitivity by incorporating Au-NPs. To further improve the SERS ability, the analyte solution was mixed with an Au-NP suspension solution in a 1:1 volume ratio before dropping it onto the substrate. As discussed previously, after a mixed droplet completely evaporating, the target analytes and Au-NPs were concentrated on the Ag-NPC. Then, thanks to the cooperation between the AuNPs and Ag nanoplates, a much higher SERS responsiveness could be achieved, and R6G was detected down to 1 pM. To prove the Ag nanoplates’ role in capturing the Au-NPs, 5 μL of the analyte and Au-NP mixture was dropped onto the thiol-decorated bare Cu substrate and allowed to evaporate. As shown in Figure S8, only a few Au-NPs were spread on the surface of the center part, different from the distribution conditions with Ag nanoplates. Thus, the hydrophilic Ag nanoplates played an important role both in capturing Au-NPs and improving the SERS response. Details on the influence of Ag-NPCs on the distribution of Au-NPs are discussed in Figure S9. 2.7. Detecting Trace Levels of Antibiotics on the SERS platform Based on the advantages discussed in the previous section, to meet the need for rapid screening for antibiotic pollutants in the environment, we were motivated to use the pre10
concentration SERS substrate to detect antibiotics detection. Thus, after mixing with Au-NPs, 5 μL of common antibiotics 6-aminopenicillanic acid (6-AA) and penicillin G sodium (PG) was dropped onto the substrate. As shown in Figure 5a and 5b, after pre-concentration via evaporation, trace concentrations of analytes could be detected. The SERS spectrum of 100 pM 6-AA exhibited dominant Raman bands at 582 and 1112 cm-1, which were assigned to ring and -CH deformations and N-N stretching, respectively (Figure 5a). A linear relationship was found between the intensities of the fingerprint peaks at 1112 cm-1 and the logarithmic concentrations of 6-AA (Figure S10a). After pre-concentration, the characteristic Raman active vibration of the phenyl ring at 1000 cm -1 was observed in the spectrum of PG at concentrations as low as 50 pM (Figure 5b). Similarly, a linear relationship was also found between the intensities of the fingerprint peaks at 1000 cm-1 and the logarithmic concentrations of PG (Figure S10b). This value is lower than that using silver-decorated silica nanocomposite rods as SERS substrates, whose detection limit for PG was found to be 100 pM.[38] To better understand of the SERS ability of our in-situ pre-concentration SERS substrate, Ag-nanoplate-assembled nanotube arrays were prepared as a substrate for comparison, as these arrays have been proven to have high-density three-dimensional SERS “hot spots”due to their high density of Ag nanoplates in 3-dimensional space (Figure S11).[39] Even so, because the surface of Ag-nanoplate-assembled nanotube arrays is hydrophilic, the analyte solution will evenly disperse on the substrate after the solution is dropped onto the surface. Then, the analyte molecules will be captured by the Ag-nanoplate-assembled nanotubes via chemi- or physi-sorption without any localized pre-concentration. Conversely, after the PG and Au-NP mixture is dropped onto the Ag-NPC-decorated Cu substrate, the Au-NPs and analyte molecules will be pre-concentrated onto a small area. Thus, as shown in two typical SERS spectra (Figure 5c) and the comparison of the average signal intensities at 1000 cm -1 (Figure 5d), the signal intensity taken from Ag-NPC/Au-NPs is almost 3 times higher than the signal 11
intensity taken from Ag-nanoplate-assembled nanotube arrays, showing the advantage of the pre-concentration substrate. At the same time, this simple but effective in-situ pre-concentration SERS substrate is comparable to the standard SERS sensor, which uses an Ag-NP-decorated graphene electrophoretic pre-concentration electrode as the SERS substrate. Its detection limits for 6AA and PG were found to be 0.7 nM and 0.3 nM, respectively.[40] Thus, this SERS substrate is promising for detecting trace-level organic pollutants in the environment. However, as the pre-concentration step on the Ag-NPC-decorated Cu substrate depends on a physical process (i.e., evaporation), the analytes are concentrated without selectivity. The selectivity will hopefully be improved by further specific modifications to the substrates. 3. Conclusions In summary, we developed a novel and simple SERS detection platform using an inkjet printer that produced a matrix pattern of AgNO3 droplets on a Cu substrate. The replacement reaction between the AgNO3 droplets and Cu not only created Ag micro-/nanostructure clusters but also selectively changed the wettability of the droplet-injected areas (from hydrophobic to hydrophilic). Subsequently, the hydrophobicity gradients of the processed Cu substrate enabled the pre-concentration of aqueous solutions via natural evaporation. For analyte detection, an Au-NP suspension solution was mixed with a target analyte solution, and 5 μL of the mixture was then dropped onto the functionalized Cu substrate. The mixture droplet required approximately 45 min to completely evaporate, leaving the target analytes surrounding the Ag-NPCs. Using this procedure, we demonstrated highly sensitive SERS detection in which two antibiotics, 6-AA and PG, were successfully detected at very low concentrations below 100 pM, showing superior performance compared to previous that of other SERS substrates such as Ag-nanoplate-assembled nanotubes. Moreover, the SERS detection platform shows many advantages such as the low cost of fabrication, high sensitivity, very small sample volumes (5 μL), and high compatibility with other microfluidic 12
components. Therefore, this platform could provide a promising and powerful means for monitoring environmental pollutants, ensuring food safety, and early warning for the presence of toxicants. 4. Experimental 4.1. Reagents and materials Cu substrates were purchased from the Goodfellow company (Huntingdon, UK). Au-NPs were purchased from Plasma Chem Company (PL-Au-S20-5 mg, 20 nm, Berlin, Germany). DMF, n-octadecanethiol, AgNO3, R6G, 6-AA and PG were purchased from Sigma Aldrich. Milli-Q deionized (DI) water (resistivity of 18.2 MΩ cm−1) was used for all solution preparations. All chemicals were used without further purification. 4.2. Surface Modification of Cu substrates and inkjet printing First, each Cu substrate was washed sequentially with acetone (10 mL), ethanol (10 mL), and DI water (100 mL). After drying, the Cu substrate was immersed in 50 mL of a 4 mM thiol DMF solution for 24 h at room temperature. The Cu substrate was then thoroughly cleaned with anhydrous ethanol to remove any residual thiol and dried in a drying oven at 60 ºC for 2 h. The piezoelectric drop-on-demand inkjet printer employed in all experiments was manufactured by Fujifilm Dimatix, Inc. (DMP-2800, CA, USA) with a cartridge (DMC11610) that supports 10 pL droplets. The printer head consists of 16 nozzles in a row with a 254 μm spacing distance. Each nozzle is approximately 21.5 μm in diameter and can be controlled individually. The center-to-center drop spacing is adjustable in one-micron increments within a range of 5 µm to 254 μm and is dependent upon the dpi setting. Arrays of droplets were printed in squares to pattern the Cu substrates with several 2 mm × 2 mm squares (Figure 3c). 4.3. Fabrication of Ag-nanoplate-assembled nanotube arrays As a control SERS substrate, nanotube arrays assembled from Ag nanoplates were fabricated using the method reported in a previous study.[39] In short, vertically aligned ZnO13
nanotaper arrays were electrodeposited on a Si substrate in a Zn(NH 3)4(NO3)2 aqueous solution using a Pt wire as the working electrode. Subsequently, Au-NPs were sputtered onto the ZnO-nanotapers to provide nucleation sites for the growth of metal building-blocks. Next, metal building blocks were assembled on the Au-NP-decorated ZnO-nanotapers via electrodeposition in an AgNO3 and citric acid electrolyte, where the electrodeposited metal grew into Ag-nanoplates under the guidance of citric acid. At the same time, the ZnOnanotapers were gradually eroded by the citric acid in the electrolyte. At the end, an array of nanotubes with their top ends closed was assembled, and noble-metal-building-blocks were achieved. 4.4. Preparation of analyte samples To investigate the SERS capabilities of the Ag-NPC-decorated Cu substrate fabricated with different concentrations of AgNO3, 1 μM R6G was used. Then, 5 μL of a 1:1 mixture of 2 μM R6G and Au-NPs was dripped onto the Ag-NPC-decorated Cu substrate to compare the SERS activity measured on Ag-NPC/Au-NPs with that of Au-NPs on the Cu surface. To detect different concentrations of 6-AA and PG, different concentrations of the antibiotics were mixed with Au-NPs in a volume ratio of 1:1. Then, 5 μL of each mixture was dripped onto the substrates. To compare the SERS activity of our in-situ pre-concentration SERS substrate with that of the Ag-nanoplate-assembled nanotube arrays, 5 μL of a PG (10 nM) and Au-NP mixture was dripped onto the Ag-NPC-decorated Cu substrate, and 5 μL PG (10 nM) solution was dripped onto the Ag-nanoplate-assembled nanotube arrays. 4.5. Experimental setup and data analysis The morphology of the resultant products was characterized by field-emission SEM (Hitachi S-4800) and EDS (Oxford). The CA of various substrates was measured using a liquid-droplet analysis tool (SmartDrop, Femtofab Co., Ltd., Korea), and the volume of the liquid droplet was 5 μL. The SERS spectra were recorded with a WITEC alpha300R microRaman system (Alphatech, New Zealand). The excitation wavelength was 532 nm from an 14
air-cooled argon ion laser with an effective power of 0.2 mW. Micrographs were taken using a high-resolution CCD camera (Eclipse 80i, Nikon, Japan).
Supporting Information Supporting Information is available.
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2014R1A2A1A10050431). This work also supported by a grant from the Next-Generation BioGreen 21 program (SSAC, PJ01118601), Rural Development Administration, Republic of Korea.
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Qitao Zhou received his B.S. degree from School of resources processing and Bioengineering, Central South University. Then he received his Ph.D. degree in Material Physics and Chemistry from University of Chinese Academy of Sciences in 2015. He then joined Professor Taesung Kim’s lab at the Ulsan National Institute of Science and Technology as a postdoctoral researcher. His research interests include nanomaterials and surfaceenhanced Raman scattering (SERS) based microfluidic sensors. Currently, he focuses on photodetector fabrication based on crack-photolithography technique.
Ashish Kumar Thokchom received his B-tech degree in Chemical Engineering from Kerala University. He received his Master degree in Industrial Pollution Control from the Department of Chemical Engineerring, NITK Surathkal, India. He received his PhD degree from Indian Institute of Technology Guwahati. He then joined Professor Taesung Kim’s group at Ulsan National Institute of Science and Technology (UNIST) as a postdoctoral researcher. His research of interests includes micro fluid flow and particle transport in micro droplet including numerical simulation, inkjet printing based micro-/nanofabrication. Currently, he focuses on the single cell trapping and electrokinetics in microfluidic device based on crack-photolithography technique.
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Dong-Joo Kim is a postdoctoral researcher in the Department of Biomedical Engineering, Yale University. He received his B.S., M.S., and Ph. D. degree from Department of Semiconductor Science and Technology, Chonbuk National University. He has developed the nanostructure based platform for separating specific target cells including circulating tumor cells (CTCs). He then joined Professor Taesung Kim’s group at Ulsan National Institute of Science and Technology (UNIST) as a postdoctoral researcher, and developed the unconventional multi-scale fabrication techniques using crack-photolithography. Currently, he focuses on the analysis of secreted proteins from single cell.
Taesung Kim is an associate professor in the Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST). He received his B.S. in 1997 and M.S. in 1999 from the Department of Mechanical Engineering, Seoul National University, and obtained his Ph.D. in 2006 from the Department of Mechanical Engineering, University of Michigan at Ann Arbor. After being a post-doctoral research fellow at the University of California at Berkeley and Lawrence Berkeley National Laboratory (JBEI), he started his independent research career at UNIST from 2009. His research interests include micro/nanofluidics, electrokinetics including numerical simulations, nanobiosensors, and inkjet printing based micro-/nanofabrication. Currently, he focuses on the development of unconventional multi-scale fabrication techniques using crack-photolithography and inkjet printing, and various microfluidic systems for bioreactors and high-throughput screening.
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Figures
Figure 1. a) Schematic showing the inkjet-printing-based fabrication process of an Ag-NPC array on an n-octadecanethiol-functionalized superhydrophobic Cu substrate. The inset shows a 5 μL water droplet on the bare Cu surface (CA = 97°). b) SEM image of the bare Cu substrate after thiol modification. The insets show the thiol-modified Cu substrate (left) and a 5 μL water droplet on the surface (right, CA = 155°). c) SEM image of the thiol-modified Cu substrate after printing with 10 pL AgNO3 droplets with a constant center-to-center spacing distance of 40 μm. The insets show an Ag-NPC-decorated superhydrophobic Cu substrate (left) and a 5 μL water droplet on the Cu surface (right, CA = 151°).
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Figure 2. a) – e) SEM images of various Ag-NPCs synthesized on the Cu substrate, which were fabricated from AgNO3 solutions with concentrations of 0.5 M, 0.25 M, 0.1 M, 0.05 M, and 0.01 M, respectively. f) The SERS response of the Ag-NPC-decorated Cu substrates fabricated with various concentrations of AgNO3 solution. g) The average SERS signal intensities of the band at 612 cm-1 for the Ag-NPC-decorated Cu substrates fabricated with various concentrations of AgNO3 solution. Each error bar was calculated from 10 independent measurements (average ± std).
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Figure 3. a) Volume changes of a water droplet as it dries on the surface. Scale bars are 1 mm. b) Schematic illustration showing the pre-concentration process of a droplet containing analytes on the substrate. c) Optical image of three types of colored 5 μL droplets containing different food dye solutions. The droplets were dripped onto the 2 mm × 2 mm Ag-NPCdecorated areas in a row, which were patterned on a 2 cm × 2 cm Cu substrate.
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Figure 4. a) SEM images of a Ag-NPC-decorated Cu substrate after a drying a 5 μL droplet of a mixture containing an analyte (2.5 μL) and the Au-NPs (2.5 μL) on the surface. (i) An enlarged image shows a coffee ring structure made of Au-NPs. Three local spots: (ii) outside the coffee ring, (iii) on the coffee ring, and (iv) inside the coffee ring. b) Schematic image showing Ag-NPC-decorated areas with Au-NPs and bare (undecorated) areas with Au-NPs on the Cu surface. c) Two typical SERS spectra taken from Ag-NPC/Au-NPs (black) and AuNPs on the Cu surface as a control (red). d) A comparison of the average signal intensities at 612 cm-1 taken from six randomly chosen spots from Ag-NPC/Au-NPs (black) and Au-NPs (red) on the Cu surface.
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Figure 5. SERS detection results for two antibiotics using the integrated SERS platform. a) SERS spectra of different concentrations of 6-AA. b) SERS spectra of different concentrations of PG. c) Two typical SERS spectra taken from Ag-NPC/Au-NPs and Agnanoplate-assembled nanotubes as a control. d) A comparison of the average signal intensities at 1000 cm-1 taken from ten randomly chosen spots of Ag-NPC/Au-NPs and Ag-nanoplateassembled nanotubes.
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S0925-4005(16)31932-3 http://dx.doi.org/doi:10.1016/j.snb.2016.11.134 SNB 21344
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
16-9-2016 17-11-2016 24-11-2016
Please cite this article as: Qitao Zhou, Ashish Kumar Thokchom, Dong-Joo Kim, Taesung Kim, Inkjet-printed Ag Micro-/Nanostructure Clusters on Cu Substrates for Insitu Pre-concentration and Surface-Enhanced Raman Scattering, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.11.134 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inkjet-printed Ag Micro-/Nanostructure Clusters on Cu Substrates for In-situ Pre-concentration and Surface-Enhanced Raman Scattering Qitao Zhou, Ashish Kumar Thokchom, Dong-Joo Kim and Taesung Kim*
Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulsan 44919, Republic of Korea.
*Corresponding author E-mail: [email protected] Department of Mechanical Engineering Ulsan National Institute of Science and Technology (UNIST) 50 UNIST-gil, Ulsan 44919, Republic of Korea.
1
Graphical abstract Surface-enhanced Raman scattering is a promising technology for biosensing but the challenge of creating practical substrates remains. This study presents a novel inkjet printing technique for fabricating (SERS) substrates that offer both in-situ fabrication of the noble metal nanoarrays, and thus high density SERS “hot spots” but also pre-concentration of the target analyte.
Highlight:
A novel inkjet printing technique for fabricating SERS substrates is presented. In-situ fabrication of noble metal nanoarrays for high density SERS hot spots and wettability gradients for pre-concentration of target analytes. Highly sensitive SERS detection of antibiotics at very low concentrations below 100 pM.
Abstract Effective surface-enhanced Raman scattering (SERS) detection requires substrates that are typically fabricated
using
expensive,
low-throughput
and
time-consuming
micro-
/nanofabrication processes such as photolithography, electron-beam lithography and templateassisted methods. Here, a novel micro-/nanofabrication technique for fabricating SERS substrates with hydrophobicity gradients is demonstrated. An inkjet printer enables injecting an AgNO3 solution onto a thiol-functionalized superhydrophobic Cu surface, upon which Ag micro/nanostructures are generated via replacement reactions in the droplet-injected areas. When a mixed solution of target analytes and Au-nanoparticles (Au-NPs) are placed on this 2
substrate, the contact area decreases over time due to the evaporation of the solution and the hydrophobicity of the substrate. As a result, the analyte molecules and Au-NPs are delivered to the Ag micro-/nanostructure clusters, upon which the analyte and Au-NPs are simply and easily concentrated in situ. With the cooperation of Ag nanoplates and Au-NPs, two antibiotics at very low concentrations (e.g., 100 pM 6-aminopenicillanic acid and 50 pM penicillin G sodium) were successfully detected, confirming the higher SERS activity than that of Ag-nanoplate-assembled nanotube arrays or an Ag-NPs decorated graphene electrophoretic pre-concentration device. Hence, this rapid design-to-prototype method for substrates with adjustable wetting properties can be very useful for a SERS platform to detect various target analytes in biosamples.
Keywords: inkjet printing, pre-concentration, wettability, surface-enhanced Raman scattering (SERS), analyte detection
1. Introduction Surface-enhanced Raman scattering (SERS) is of particular interest for detecting and monitoring hazardous and/or harmful molecules, because it can rapidly detect analytes while simultaneously providing their mass fingerprint spectra. Widely used mass-based technologies such as gas chromatography with mass spectrometry (GC–MS),[1] or mass spectrometry (LC–MS)[2] can elucidate the chemical structures and mass fingerprints of molecules, but they usually require complex extractions and purification procedures, which are both time- and sample-consuming.[3] On the other hand, more rapid detection methods such as enzyme-linked immunosorbent assays (ELISA)[4] and fluorescent sensors[5] are 3
usually based on specific biochemical or physicochemical interactions and cannot provide mass fingerprints. For effective SERS detection, plasmonic metal nanostructures are used to amplify the sensing signal.[6-8] Thus, various noble metal nanostructures have been proposed. For example, Au and Ag colloidal nanoparticles have been used as platforms to achieve high SERS-activity from “hot spots” (i.e., sub-10-nm-gaps between the noble metal nanoparticles) in randomly dispersed colloidal systems;[9-13] however, the reproducibility of SERS signals from different locations is poor, thus restricting SERS-based practical applications. Regularly patterned arrays of noble metal nanoparticles[14] and, nanorods[15] and Ag semishells[16] were fabricated with the assistance of templates such as porous anodic aluminum oxide (AAO) or colloidal polystyrene (PS) spheres to achieve good SERS signal reproducibility with high sensitivity, as high density “hot spots” could be homogeneously distributed on the whole patterned substrate. However, these template-assisted methods usually have several impractical steps and are time consuming. Developing a template-free fabrication method for noble metal micro/nanostructure arrays has considerable practical significance. Recently, inkjet printing has been developed into an effective method for fabricating micro-/nanostructure arrays as this type of printing can simply drop the ink solution in a fixed volume onto a set position.[17-20] Based on these advantages, Ag or Au nanoparticles have been printed on cellulose paper or silicon wafers for use as SERS substrates.[21, 22] However, in these cases, the nanostructures in the inks, such as colloidal Ag nanoparticles, needed to be synthesized in advance. Therefore, inkjet printing redistributed the nanostructures rather than fabricating them, which does not fully exploit the advantages of inkjet printing, such as its simplicity and efficiency. Thus, developing an inkjet-printing-based method that offers in-suit fabrication of noble metal micro-/nanostructure would be a significant step forward. On the other hand, to enable sensitive SERS detection, as many analyte molecules as possible should be in the vicinity of the plasmonic nanostructure surface so that the SERS-active molecules 4
are exposed to the strong electromagnetic field generated by the plasmonic nanostructures.[2326]
For instance, mono-6-thio-β-cyclodextrin has been functionalized onto a SERS substrate to
capture analyte molecules such as polychlorinated biphenyls and methyl parathion. [27,
28]
However, the trapping effect of cyclodextrin depends on the design of the cyclodextrin cavity diameter,[29] which is complicated and time-consuming. Alternatively, macromolecules such as proteins, antibodies and DNA immobilized on SERS substrates have been employed to capture the antigen or analytes,[30-32] but such samples are difficult to prepare and preserve. To solve these problems, SERS substrates with a hydrophobicity gradient have been designed and prepared. This hydrophobicity gradient is intended to draw all the molecules in a sample into the detection region of the SERS substrate. For example, electron-beam lithography (EBL) and focused ion beam (FIB) milling were used to fabricate SERS-active elements and structures for superhydrophobicity. During the detection, analyte molecules in the droplet were extracted to the SERS-active Ag-decorated nanocone in the center, thus realizing SERS detection at ultra-low concentrations.[33] To make the fabrication process simpler and less expensive, optical lithography have has been used rather than EBL and FIB to fabricate a superhydrophobic bull's-eye substrate. After a water droplet containing a gold nanoparticle colloid was pipetted onto the superhydrophobic bull's-eye substrate, the gold nanoparticles aggregated at the center to act as the SERS active region.[34] Nevertheless, this approach is still not sufficiently simple or inexpensive for a ready-to-use SERS substrate. Thus, developing a SERS substrate with a hydrophobicity gradient using a template-free fabrication method remains a challenge. In this work, we describe a novel, simple and convenient method to fabricate metal micro/nanostructure clusters using an inkjet printer. We optimize the concentration of a silver nitrate (AgNO3) solution as an ink, and we then characterize the Ag micro-/nanofabrication process in which Ag nanoplate clusters (Ag-NPCs) are created via the substitution reaction between Cu and AgNO3.[35] Furthermore, we characterize the surface wettability effect by 5
adjusting the spacing distance between the Ag-NPCs, thus functionalizing the Cu surface to endow it with a hydrophobicity gradient. Recently, the development of reliable analytical techniques to detect antibiotic residues in the environment has garnered considerable interest, as the needless overuse of antibiotics can lead to drug resistance, toxicity, and allergic reactions.[36] Therefore, we demonstrate a novel SERS detection platform for antibiotics by exposing the engineered hydrophobicity gradient to droplets of target analytes and Au nanoparticles (Au-NPs), which are then pre-concentrated to enhance the SERS activity. Lastly, we compare the performance of our platform with previous SERS-based detection approaches. 2. Results and Discussion 2.1. Fabrication of Ag-NPCs on thiol-decorated Cu substrates Figure 1 shows the substrate fabrication steps. Because the hydrophobicity gradient plays an important part in pre-concentrating the target analytes, we modified the surface wettability of the Cu surface using a thiol coating, and we regulated the printing conditions of AgNO3. As shown in Figure 1a, the contact angle (CA) of water on a bare Cu substrate was 97°, indicating the relatively weak hydrophobicity of the bare Cu substrate. To achieve a superhydrophobic surface, we functionalized the substrate in n-octadecanethiol solution in N,N-dimethylformamide (DMF), as described below in the Experimental section. The low surface energy of the surface bonded n-octadecanethiol significantly increased the hydrophobicity of the bare Cu substrate. The morphology of the Ag-NPCs synthesized via inkjet printing was measured using scanning electron microscopy (SEM). As shown in Figure 1b, thiol modification increased the CA to 155°. Then, a AgNO3 solution was injected using the inkjet printer onto the superhydrophobic Cu surface. As Cu has higher reactivity, the Ag ions were reduced to Ag nanoplates, and the Ag nanoplates grew from the Cu surface, as shown in Figure 1c. In turn, regular Ag-NPCs composed of interlaced Ag nanoplates formed on the Cu substrate surface. The formation of metallic Ag was confirmed by energy dispersive X-ray spectroscopy (EDS) (Figure S1 in the Supporting Information). 6
2.2. Effect of print spacing on the wettability of Ag-NPC arrays After printing on the designated areas of the thiol-decorated Cu substrate, the hydrophobicity of these areas will be reduced. The inset in Figure 1c shows that the CA changed from 155° to 151°due to these Ag-NPC arrays. In order to demonstrate the role of Ag-NPC arrays in regulating the wettability of the surface, the bare Cu substrate have was printed with Ag micro-/nanostructure arrays at various distances between the centers of neighboring droplets. The CA could be adjusted from 67° to 53° as the distances between the arrays were decreased from 120 μm to 60 μm and 40 μm (Figure S2). As the distance between the hydrophilic Ag clusters decreased, the amount of the Cu surface covered by the clusters (i.e., the surface density of the clusters increased), thus decreasing the CA. Therefore, the AgNPCs can increase the hydrophilicity of the surface. Similarly, a region with lower hydrophobicity can be obtained in the droplet printed areas on the superhydrophobic Cu surface. To achieve a steeper hydrophobicity gradient and to ensure that the AgNO3 droplets would not interact with each other, 40 μm was set as the printing distance in the subsequent experiments. Interestingly, if the thiol-treatment step was implemented after inkjet printing, the hydrophobicity of the Cu substrate could be further improved (Figure S3). The obvious increase in the CA (from 151° to 168°) was due to the increasing amount of surface area with a low surface energy. This increase occurred because when the Cu substrate decorated with Ag-NPCs was immersed into the thiol solution, the thiol molecules labeled both the Cu and Ag surfaces. 2.3. Effect of AgNO3 concentration on SERS activity As shown in Figure 2, the size and SERS activity of the Ag nanoplates can be tuned by adjusting the concentration of AgNO3. With the decrease in AgNO3 concentration from 0.5 M to 0.1 M, the thickness of the Ag nanoplates decreased, but the density of the Ag nanoplates increased, leading to smaller gaps and the number increase of nano-gaps (less than 10 nm, i.e., the desired so-called “hot spots”) increased between nearest neighboring Ag nanoplates. 7
Therefore, more “hot spots” were achieved, which improved the SERS activity (Figure 2a-e). However, with a further decrease in the AgNO3 concentration, the Ag nanoplates became smaller and sparser, and therefore, the “hot spots” in the small gaps decreased between neighboring Ag nanoplates, leading to a decrease in the SERS activity, as shown in the upper two spectra in Figure 2f. As shown in Figure 2g, the Ag-NPC-decorated Cu substrate prepared with 0.1 M AgNO3 showed the strongest SERS activity. Thus, it was used as the SERS substrate in the subsequent experiments. As shown in Figure S4, SERS mapping images were obtained from the five samples prepared by different AgNO3 concentrations for the objective comparison of SERS activity. It was confirmed that the signal intensities changing with the AgNO3 concentrations show the similar tendency as Figure 2g. 2.4. Pre-concentrating and fixing the analyte via evaporation Figure 3a shows the drying process of a water droplet placed on the functional Cu substrate over time. As water evaporates from the drop, its volume shrinks. However, its shape remains, and it is fixed at the same center of the substrate. During most of this drying process, the CA of the droplet remains almost constant. However, further drying eventually results in sharp reductions in the CA. When the water droplet is replaced by an analyte solution, the molecules in the solution can be concentrated into a small area, as illustrated in Figure 3b. This is beneficial to SERS detection because the higher the analyte concentration, the more sensitive the SERS detection. Not only can the hydrophobicity gradient preconcentrate the analyte, but it can also help fix the analyte droplet on the surface. Notably, the Ag-NPC-decorated Cu surface could immobilize the solution droplet at a fixed place (refer to Supplementary Movie S1). Specifically, when the substrate is tilted at an increasing angle, the solution droplet stays in the same place on the Ag-NPC-printed substrate; meanwhile, on the unprinted thiol-decorated superhydrophobic Cu substrate, the droplet slides down. This result shows the difference between the wettability of the thiol-decorated Cu surface and the Agnanoplate-cluster-decorated Cu substrate, displaying its ability to capture droplets. In addition, 8
many small droplets containing analyte molecules can be fixed on the substrate without interfering with each other, implying that different concentrations of different analytes can be detected on a single SERS substrate. As shown in Figure 3c, 5 μL droplets of different dye solutions at various concentrations were dripped one by one onto the regular 2 mm × 2 mm printed patterns on a 2 cm × 2 cm Cu substrate, and the droplets fixed onto the printed patterns very well. 2.5. Decorating the Ag-NPCs with Au-NPs As shown in Figure 4, we tested the SERS ability of the Ag-NPCs by incorporating the Au-NP solution. After evaporation, a visible ring pattern formed caused by a coffee-ring effect (Figure 4a(i)).[22, 37] Outside the ring, there are almost no Au-NPs (Figure 4a(ii)). Due to the difference between the wettability of the thiol-decorated Cu surface and that of the AgNPCs, the Au-NPs preferentially deposited on the Ag nanoplates. The SEM image taken from the area in the coffee ring (Figure 4a(iv)) shows that some Au-NP clusters were randomly distributed around the Ag-NPC. Accordingly, a large number of Au-NPs deposited on the Ag nanoplates due to their hydrophilicity. As schematically shown in Figure 4b, to compare the SERS ability of Ag-NPCs decorated by Au-NPs (Ag-NPC/Au-NPs) and Au-NPs alone on the Cu surface, six spots were randomly chosen on the substrate to measure. Comparing the average signal intensities at 612 cm-1 in the two typical SERS spectra (Figure 4c-d), the signal taken from Ag-NPCs/Au-NPs is almost 6 times more intense than that of the Au-NPs alone on the Cu surface. This higher intensity is because not only can the Ag nanoplates supply “hot spots”, but also because there are many “hot spots” between the Au-NPs. In addition, rather than dropping the analyte solution on the SERS substrate, we measured the SERS signals by immersing the substrate in the analyte solution and then drying it for comparison (see Figure S5). It turned out that the signal intensities taken from the dropped sample are at least 3 times higher than those taken from the immersed sample, verifying that the pre-concentration of the analyte driven by a hydrophobicity gradient is much stronger than the self-adsorption of the 9
analyte to the SERS substrate. Furthermore, the plasmonic coupling between the Ag nanoplates and the Au-NPs can also further improve the SERS ability. Figure S6 shows a SERS mapping image taken from the Ag-NPCs/Au-NPs on a substrate in which the central part with the cooperation of Ag-NPCs and Au-NPs exhibits much stronger and uniform SERS signals. In contrast, lower and nonuniform signals are taken from the rest part as there are only some randomly distributed Au-NPs. 2.6. SERS performance with and without Au-NPs We compared the SERS performance of an Ag-NPC-decorated Cu substrate with that of the Ag-NPC-decorated Cu substrate with Au-NPs using a well-known molecule (e.g., rhodamine 6G [R6G]), as shown in Figure S7. Although the Ag-NPC-decorated Cu substrate can successfully detected R6G down to 100 pM, we aimed to enhance the sensitivity by incorporating Au-NPs. To further improve the SERS ability, the analyte solution was mixed with an Au-NP suspension solution in a 1:1 volume ratio before dropping it onto the substrate. As discussed previously, after a mixed droplet completely evaporating, the target analytes and Au-NPs were concentrated on the Ag-NPC. Then, thanks to the cooperation between the AuNPs and Ag nanoplates, a much higher SERS responsiveness could be achieved, and R6G was detected down to 1 pM. To prove the Ag nanoplates’ role in capturing the Au-NPs, 5 μL of the analyte and Au-NP mixture was dropped onto the thiol-decorated bare Cu substrate and allowed to evaporate. As shown in Figure S8, only a few Au-NPs were spread on the surface of the center part, different from the distribution conditions with Ag nanoplates. Thus, the hydrophilic Ag nanoplates played an important role both in capturing Au-NPs and improving the SERS response. Details on the influence of Ag-NPCs on the distribution of Au-NPs are discussed in Figure S9. 2.7. Detecting Trace Levels of Antibiotics on the SERS platform Based on the advantages discussed in the previous section, to meet the need for rapid screening for antibiotic pollutants in the environment, we were motivated to use the pre10
concentration SERS substrate to detect antibiotics detection. Thus, after mixing with Au-NPs, 5 μL of common antibiotics 6-aminopenicillanic acid (6-AA) and penicillin G sodium (PG) was dropped onto the substrate. As shown in Figure 5a and 5b, after pre-concentration via evaporation, trace concentrations of analytes could be detected. The SERS spectrum of 100 pM 6-AA exhibited dominant Raman bands at 582 and 1112 cm-1, which were assigned to ring and -CH deformations and N-N stretching, respectively (Figure 5a). A linear relationship was found between the intensities of the fingerprint peaks at 1112 cm-1 and the logarithmic concentrations of 6-AA (Figure S10a). After pre-concentration, the characteristic Raman active vibration of the phenyl ring at 1000 cm -1 was observed in the spectrum of PG at concentrations as low as 50 pM (Figure 5b). Similarly, a linear relationship was also found between the intensities of the fingerprint peaks at 1000 cm-1 and the logarithmic concentrations of PG (Figure S10b). This value is lower than that using silver-decorated silica nanocomposite rods as SERS substrates, whose detection limit for PG was found to be 100 pM.[38] To better understand of the SERS ability of our in-situ pre-concentration SERS substrate, Ag-nanoplate-assembled nanotube arrays were prepared as a substrate for comparison, as these arrays have been proven to have high-density three-dimensional SERS “hot spots”due to their high density of Ag nanoplates in 3-dimensional space (Figure S11).[39] Even so, because the surface of Ag-nanoplate-assembled nanotube arrays is hydrophilic, the analyte solution will evenly disperse on the substrate after the solution is dropped onto the surface. Then, the analyte molecules will be captured by the Ag-nanoplate-assembled nanotubes via chemi- or physi-sorption without any localized pre-concentration. Conversely, after the PG and Au-NP mixture is dropped onto the Ag-NPC-decorated Cu substrate, the Au-NPs and analyte molecules will be pre-concentrated onto a small area. Thus, as shown in two typical SERS spectra (Figure 5c) and the comparison of the average signal intensities at 1000 cm -1 (Figure 5d), the signal intensity taken from Ag-NPC/Au-NPs is almost 3 times higher than the signal 11
intensity taken from Ag-nanoplate-assembled nanotube arrays, showing the advantage of the pre-concentration substrate. At the same time, this simple but effective in-situ pre-concentration SERS substrate is comparable to the standard SERS sensor, which uses an Ag-NP-decorated graphene electrophoretic pre-concentration electrode as the SERS substrate. Its detection limits for 6AA and PG were found to be 0.7 nM and 0.3 nM, respectively.[40] Thus, this SERS substrate is promising for detecting trace-level organic pollutants in the environment. However, as the pre-concentration step on the Ag-NPC-decorated Cu substrate depends on a physical process (i.e., evaporation), the analytes are concentrated without selectivity. The selectivity will hopefully be improved by further specific modifications to the substrates. 3. Conclusions In summary, we developed a novel and simple SERS detection platform using an inkjet printer that produced a matrix pattern of AgNO3 droplets on a Cu substrate. The replacement reaction between the AgNO3 droplets and Cu not only created Ag micro-/nanostructure clusters but also selectively changed the wettability of the droplet-injected areas (from hydrophobic to hydrophilic). Subsequently, the hydrophobicity gradients of the processed Cu substrate enabled the pre-concentration of aqueous solutions via natural evaporation. For analyte detection, an Au-NP suspension solution was mixed with a target analyte solution, and 5 μL of the mixture was then dropped onto the functionalized Cu substrate. The mixture droplet required approximately 45 min to completely evaporate, leaving the target analytes surrounding the Ag-NPCs. Using this procedure, we demonstrated highly sensitive SERS detection in which two antibiotics, 6-AA and PG, were successfully detected at very low concentrations below 100 pM, showing superior performance compared to previous that of other SERS substrates such as Ag-nanoplate-assembled nanotubes. Moreover, the SERS detection platform shows many advantages such as the low cost of fabrication, high sensitivity, very small sample volumes (5 μL), and high compatibility with other microfluidic 12
components. Therefore, this platform could provide a promising and powerful means for monitoring environmental pollutants, ensuring food safety, and early warning for the presence of toxicants. 4. Experimental 4.1. Reagents and materials Cu substrates were purchased from the Goodfellow company (Huntingdon, UK). Au-NPs were purchased from Plasma Chem Company (PL-Au-S20-5 mg, 20 nm, Berlin, Germany). DMF, n-octadecanethiol, AgNO3, R6G, 6-AA and PG were purchased from Sigma Aldrich. Milli-Q deionized (DI) water (resistivity of 18.2 MΩ cm−1) was used for all solution preparations. All chemicals were used without further purification. 4.2. Surface Modification of Cu substrates and inkjet printing First, each Cu substrate was washed sequentially with acetone (10 mL), ethanol (10 mL), and DI water (100 mL). After drying, the Cu substrate was immersed in 50 mL of a 4 mM thiol DMF solution for 24 h at room temperature. The Cu substrate was then thoroughly cleaned with anhydrous ethanol to remove any residual thiol and dried in a drying oven at 60 ºC for 2 h. The piezoelectric drop-on-demand inkjet printer employed in all experiments was manufactured by Fujifilm Dimatix, Inc. (DMP-2800, CA, USA) with a cartridge (DMC11610) that supports 10 pL droplets. The printer head consists of 16 nozzles in a row with a 254 μm spacing distance. Each nozzle is approximately 21.5 μm in diameter and can be controlled individually. The center-to-center drop spacing is adjustable in one-micron increments within a range of 5 µm to 254 μm and is dependent upon the dpi setting. Arrays of droplets were printed in squares to pattern the Cu substrates with several 2 mm × 2 mm squares (Figure 3c). 4.3. Fabrication of Ag-nanoplate-assembled nanotube arrays As a control SERS substrate, nanotube arrays assembled from Ag nanoplates were fabricated using the method reported in a previous study.[39] In short, vertically aligned ZnO13
nanotaper arrays were electrodeposited on a Si substrate in a Zn(NH 3)4(NO3)2 aqueous solution using a Pt wire as the working electrode. Subsequently, Au-NPs were sputtered onto the ZnO-nanotapers to provide nucleation sites for the growth of metal building-blocks. Next, metal building blocks were assembled on the Au-NP-decorated ZnO-nanotapers via electrodeposition in an AgNO3 and citric acid electrolyte, where the electrodeposited metal grew into Ag-nanoplates under the guidance of citric acid. At the same time, the ZnOnanotapers were gradually eroded by the citric acid in the electrolyte. At the end, an array of nanotubes with their top ends closed was assembled, and noble-metal-building-blocks were achieved. 4.4. Preparation of analyte samples To investigate the SERS capabilities of the Ag-NPC-decorated Cu substrate fabricated with different concentrations of AgNO3, 1 μM R6G was used. Then, 5 μL of a 1:1 mixture of 2 μM R6G and Au-NPs was dripped onto the Ag-NPC-decorated Cu substrate to compare the SERS activity measured on Ag-NPC/Au-NPs with that of Au-NPs on the Cu surface. To detect different concentrations of 6-AA and PG, different concentrations of the antibiotics were mixed with Au-NPs in a volume ratio of 1:1. Then, 5 μL of each mixture was dripped onto the substrates. To compare the SERS activity of our in-situ pre-concentration SERS substrate with that of the Ag-nanoplate-assembled nanotube arrays, 5 μL of a PG (10 nM) and Au-NP mixture was dripped onto the Ag-NPC-decorated Cu substrate, and 5 μL PG (10 nM) solution was dripped onto the Ag-nanoplate-assembled nanotube arrays. 4.5. Experimental setup and data analysis The morphology of the resultant products was characterized by field-emission SEM (Hitachi S-4800) and EDS (Oxford). The CA of various substrates was measured using a liquid-droplet analysis tool (SmartDrop, Femtofab Co., Ltd., Korea), and the volume of the liquid droplet was 5 μL. The SERS spectra were recorded with a WITEC alpha300R microRaman system (Alphatech, New Zealand). The excitation wavelength was 532 nm from an 14
air-cooled argon ion laser with an effective power of 0.2 mW. Micrographs were taken using a high-resolution CCD camera (Eclipse 80i, Nikon, Japan).
Supporting Information Supporting Information is available.
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2014R1A2A1A10050431). This work also supported by a grant from the Next-Generation BioGreen 21 program (SSAC, PJ01118601), Rural Development Administration, Republic of Korea.
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Qitao Zhou received his B.S. degree from School of resources processing and Bioengineering, Central South University. Then he received his Ph.D. degree in Material Physics and Chemistry from University of Chinese Academy of Sciences in 2015. He then joined Professor Taesung Kim’s lab at the Ulsan National Institute of Science and Technology as a postdoctoral researcher. His research interests include nanomaterials and surfaceenhanced Raman scattering (SERS) based microfluidic sensors. Currently, he focuses on photodetector fabrication based on crack-photolithography technique.
Ashish Kumar Thokchom received his B-tech degree in Chemical Engineering from Kerala University. He received his Master degree in Industrial Pollution Control from the Department of Chemical Engineerring, NITK Surathkal, India. He received his PhD degree from Indian Institute of Technology Guwahati. He then joined Professor Taesung Kim’s group at Ulsan National Institute of Science and Technology (UNIST) as a postdoctoral researcher. His research of interests includes micro fluid flow and particle transport in micro droplet including numerical simulation, inkjet printing based micro-/nanofabrication. Currently, he focuses on the single cell trapping and electrokinetics in microfluidic device based on crack-photolithography technique.
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Dong-Joo Kim is a postdoctoral researcher in the Department of Biomedical Engineering, Yale University. He received his B.S., M.S., and Ph. D. degree from Department of Semiconductor Science and Technology, Chonbuk National University. He has developed the nanostructure based platform for separating specific target cells including circulating tumor cells (CTCs). He then joined Professor Taesung Kim’s group at Ulsan National Institute of Science and Technology (UNIST) as a postdoctoral researcher, and developed the unconventional multi-scale fabrication techniques using crack-photolithography. Currently, he focuses on the analysis of secreted proteins from single cell.
Taesung Kim is an associate professor in the Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST). He received his B.S. in 1997 and M.S. in 1999 from the Department of Mechanical Engineering, Seoul National University, and obtained his Ph.D. in 2006 from the Department of Mechanical Engineering, University of Michigan at Ann Arbor. After being a post-doctoral research fellow at the University of California at Berkeley and Lawrence Berkeley National Laboratory (JBEI), he started his independent research career at UNIST from 2009. His research interests include micro/nanofluidics, electrokinetics including numerical simulations, nanobiosensors, and inkjet printing based micro-/nanofabrication. Currently, he focuses on the development of unconventional multi-scale fabrication techniques using crack-photolithography and inkjet printing, and various microfluidic systems for bioreactors and high-throughput screening.
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Figures
Figure 1. a) Schematic showing the inkjet-printing-based fabrication process of an Ag-NPC array on an n-octadecanethiol-functionalized superhydrophobic Cu substrate. The inset shows a 5 μL water droplet on the bare Cu surface (CA = 97°). b) SEM image of the bare Cu substrate after thiol modification. The insets show the thiol-modified Cu substrate (left) and a 5 μL water droplet on the surface (right, CA = 155°). c) SEM image of the thiol-modified Cu substrate after printing with 10 pL AgNO3 droplets with a constant center-to-center spacing distance of 40 μm. The insets show an Ag-NPC-decorated superhydrophobic Cu substrate (left) and a 5 μL water droplet on the Cu surface (right, CA = 151°).
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Figure 2. a) – e) SEM images of various Ag-NPCs synthesized on the Cu substrate, which were fabricated from AgNO3 solutions with concentrations of 0.5 M, 0.25 M, 0.1 M, 0.05 M, and 0.01 M, respectively. f) The SERS response of the Ag-NPC-decorated Cu substrates fabricated with various concentrations of AgNO3 solution. g) The average SERS signal intensities of the band at 612 cm-1 for the Ag-NPC-decorated Cu substrates fabricated with various concentrations of AgNO3 solution. Each error bar was calculated from 10 independent measurements (average ± std).
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Figure 3. a) Volume changes of a water droplet as it dries on the surface. Scale bars are 1 mm. b) Schematic illustration showing the pre-concentration process of a droplet containing analytes on the substrate. c) Optical image of three types of colored 5 μL droplets containing different food dye solutions. The droplets were dripped onto the 2 mm × 2 mm Ag-NPCdecorated areas in a row, which were patterned on a 2 cm × 2 cm Cu substrate.
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Figure 4. a) SEM images of a Ag-NPC-decorated Cu substrate after a drying a 5 μL droplet of a mixture containing an analyte (2.5 μL) and the Au-NPs (2.5 μL) on the surface. (i) An enlarged image shows a coffee ring structure made of Au-NPs. Three local spots: (ii) outside the coffee ring, (iii) on the coffee ring, and (iv) inside the coffee ring. b) Schematic image showing Ag-NPC-decorated areas with Au-NPs and bare (undecorated) areas with Au-NPs on the Cu surface. c) Two typical SERS spectra taken from Ag-NPC/Au-NPs (black) and AuNPs on the Cu surface as a control (red). d) A comparison of the average signal intensities at 612 cm-1 taken from six randomly chosen spots from Ag-NPC/Au-NPs (black) and Au-NPs (red) on the Cu surface.
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Figure 5. SERS detection results for two antibiotics using the integrated SERS platform. a) SERS spectra of different concentrations of 6-AA. b) SERS spectra of different concentrations of PG. c) Two typical SERS spectra taken from Ag-NPC/Au-NPs and Agnanoplate-assembled nanotubes as a control. d) A comparison of the average signal intensities at 1000 cm-1 taken from ten randomly chosen spots of Ag-NPC/Au-NPs and Ag-nanoplateassembled nanotubes.
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