先進二維光電科技研究 - 石墨烯之合成、光電效應、及新穎元件之探討

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(1)科技部補助專題研究計畫報告. 先進二維光電科技研究 - 石墨烯之合成、光電效應、及新穎元件之探討(第3年). 報計計執執. 告畫畫行行. 類類編期單. 別別號間位. ：：：：：. 成果報告個別型計畫 MOST 106-2221-E-006-173-MY3 108年08月01日至109年07月31日國立成功大學微電子工程研究所. 計畫主持人：曾永華. 計畫參與人員：碩士班研究生-兼任助理：黃博彥碩士班研究生-兼任助理：陳子洋碩士班研究生-兼任助理：毛彥翔碩士班研究生-兼任助理：林柏儀碩士班研究生-兼任助理：何佳霖碩士班研究生-兼任助理：沈開一. 本研究具有政策應用參考價值：■否 □是，建議提供機關（勾選「是」者，請列舉建議可提供施政參考之業務主管機關）本研究具影響公共利益之重大發現：□否 □是 . 中華民國 109 年 10 月 28 日.

(2) 中文摘要：二維奈米材料和結構呈現出一些獨特且優秀的光、電、磁、熱、化學等特性，是科學家、工程師和研究生們進一步發現科學新知和構想、設計、製作新穎光電元件的極佳機會。純單層石墨烯的能隙是零，其電子與電洞在適當條件下的等效質量也是零，因此他們的行為就像相對粒子一樣，以超高速度彈道傳輸，並呈現熱電子及多重離子化激發電子-電洞對的現象。石墨烯雖僅有一個原子的厚度，卻能吸收2.3%的白光，具有飽和光吸現像，而且其導電度對表面及近距離電荷敏感。本計劃研究依石墨烯表面沈積特性，合成製造奈米高敏感度光電感測器。依微-奈米級石墨烯之結構而合成的金屬奈米表面增強拉曼散射元件及場效電晶體，適合發展成極敏感的感測器。主持人研究團隊深具石墨烯合成及元件製造經驗。主持人研究團隊，獲得1 項發明專利(US patent #10,429,308) 並發表四篇期刊論文。中文關鍵詞：二維材料、奈米科技、石墨烯、感測元件英文摘要： Monolayer graphene is ultra-thin and optically and electromagnetically transparent in a wide energy range. Its perfect 2-D sp2 carbon-carbon bonding result in outstanding electronic, thermal, magnetic, and photonic properties which are very much desirable for scientific and commercial applications. New and excellent optoelectronic effects, on which novel devices are based will be discovered and investigated. In the recent one year, selective deposition of closely packed yet separated silver nanoparticles have been demonstrated using graphene nanoscale islands as a template. The silver nanostructure exhibits excellent surface enhanced Raman scattering signal strength measured from molecules of as low concentration as 10-16 M. Diamond NV center is based on for a new field effect transistor to operate under multi-wavelength optical inputs. A US patent (US patent #10,429,308, Oct. 2019) was granted. Four refereed journal papers have been published. 英文關鍵詞： Graphene, 2-D material, SERS, NV Center, FET.

(3) (三) (Report) 報告內容 In this report, brief introduction is first provided. This is followed by four published papers, which describe details of the technical contents.. (1) Background 前言 Monolayer graphene is ultra-thin and optically and electromagnetically transparent in a wide energy range. Its perfect 2-D sp2 carbon-carbon bonding result in outstanding electronic, thermal, magnetic, and photonic properties which are very much desirable for scientific and commercial applications. New and excellent optoelectronic effects, on which novel devices are based, have been investigated with new inventions and new knowledge having been discovered.. (2) Objectives 研究目的 Novel approaches to the synthesis of graphene of unique properties and its applications to advanced fabrication processes and high performance optoelectronic devices are pursued for promoting economy and improving human life. For advanced materials and devices, graphene was applied to serve (I) as a template for selective deposition of nanostructured silver crystals for SERS sensors; and (II) as a charge sensitive conductor for an optically driven FET based on graphene sensing of charge state of diamond NV center.. (3) Technical Approach 研究方法 (3)-1 SERS Achieving the very low detection limit of graphene-silver SERS to as low as 10-16 M R6G and 10-12 M adenine reproducibly was made possible by optimizing the graphene template so that silver nanoparticles deposit only on copper surface without graphene coverage. Silver crystals are synthesized in desirable shape and size while the gap, which separates two silver nanoparticles can be optimized for the induction of the maximum local electric field by plasmonic coupling. (3)-2 Diamond NV-Center Charge State Sensitive Graphene-on-Diamond FET High quality monolayer graphene synthesized by thermal CVD is transferred to a piece of diamond to fabricate a graphene-on-diamond field-effect transistor. NV center is formed by a vacancy in diamond along with a neighboring substitutional nitrogen. 1.

(4) The NV center can be either neutral or negatively charged. Neutral and negative charge states of NV centers in diamond can be changed back and forth by illuminating the FET by light of different wavelengths. This makes it possible for the invention of a new FET with wavelength dependent multiple optical inputs and one electrical output based on the charge-sensitive conductivity of graphene.. (4) Accomplishments and Conclusion 結果與討論（含結論與建議） (4)-1. Yonhua Tzeng, Ying-Ren Chen, Jiun-Chi Lai, Boyen Huang. Silver Nanoparticles SERS Sensors Using Rapid Thermal CVD Nanoscale Graphene Islands as Templates. IEEE Transactions on Nanotechnology, Vol. 19, pp. 25-33, 2020. (4)-2. Yonhua Tzeng and Bo-Yi Lin. Silver SERS Adenine Sensors with a Very Low Detection Limit. Biosensors, 10, 53, 2020. (4)-3. Yonhua Tzeng and Bo-Yi Lin. Silver-Based SERS Pico-MolarAdenine Sensor. Biosensors, 10, 122, 2020. (Because the combined file size exceeds the maximum 10MB set by the MOST website, this paper is not included in the report but can be read at Biosensors 2020, 10(9), 122; https://doi.org/10.3390/bios10090122 ) (4)-4. Yonhua Tzeng, Fellow, Ying-Ren Chen, Pin-Yi Li, Chun-Cheng Chang, Yueh-Chieh Chu. NV Center Charge State Controlled Graphene-on-Diamond Field Effect TransistorActions with MultiWavelength Optical Inputs. IEEE Open Journal of Nanotechnology, Vol. 1, Issue 1, pp. 18-24, 2020.. 2.

(5) IEEE TRANSACTIONS ON NANOTECHNOLOGY, Volume 19, 2020. 25. Silver Nanoparticles SERS Sensors Using Rapid Thermal CVD Nanoscale Graphene Islands as Templates Yonhua Tzeng , Fellow, IEEE, YingRen Chen, JiunChi Lai, and Boyen Huang. Abstract—Surface enhanced Raman scattering (SERS) sensors have been fabricated by rapid thermal chemical vapor deposition of high-density nanoscale discrete graphene islands on copper foils followed by electroless chemical plating of discrete, closely spaced, and irregularly shaped silver nanoparticles on the copper surface where it is not covered by graphene islands. By fine tuning of the size and distribution of graphene islands and adjusting the deposition time for silver nanoparticles, nanoscale gaps between silver particles are fabricated. SERS sensors exhibiting Raman scattering signal enhancement factors as high as 1014 in reference to a bare copper have been demonstrated. Raman scattering signal has been measured from as low as 10−16 M of R6G molecules in water. This article reports effects and optimization process of size and distribution of graphene islands on desirable morphology of chemically plated silver nanoparticles. The density of nanoscale gaps of a few nanometers in distance between neighboring silver nanoparticles is optimized, resulting in the demonstration of SERS sensors with very low detection limits for R6G molecules. Index Terms—SERS, silver, graphene, rapid thermal CVD, R6G.. I. INTRODUCTION HEN closely spaced silver nanoparticles of proper sizes are illuminated by a laser beam of a proper power and wavelength, electrons are accelerated by the oscillating electric field of the laser beam and drift synchronously within individual silver nanoparticles. The drift of electrons results in high-density electrical charges of opposite signs (accumulation and depletion of free electrons) on two counter surfaces of the gap between two neighboring silver nanoparticles. The local electric fields induced by the charges of opposite signs across the nanoscale gap can be many orders of magnitude higher than the electromagnetic wave of the exciting laser beam. This is known as plasmonic coupling effects. If the nanoscale gap is formed between two surfaces of large curvatures, such as two needle-like surfaces, the local field enhancement is even higher. If a molecule is present in such a nanoscale gap, the Raman. W. Manuscript received May 19, 2019; revised August 25, 2019; accepted November 16, 2019. Date of publication November 26, 2019; date of current version December 16, 2019. This work was supported by the Ministry of Science and Technology in Taiwan under Grant MOST-106-2221-E-006-173-MY3 and Grant MOST-105-2221-E-006-057-MY3. The review of this article was arranged by Associate Editor F. Ye. (Corresponding author: Yonhua Tzeng.) The authors are with the Department of Electrical Engineering, National Cheng Kung University, Tainan 701, Taiwan (e-mail: tzengyo@gmail. com; [email protected]; [email protected]; timmy2426a@ gmail.com). Digital Object Identifier 10.1109/TNANO.2019.2954490. scattering signal strength measured from the molecule can be enhanced by many orders of magnitude compared to that without the plasmonic coupling effect. If the concentration of nanoscale gaps is so high that many molecules are adsorbed in the gaps, the Raman scattering signal to noise ratio is high enough and the characteristic Raman scattering peaks from the molecules can be detected and the molecules can be precisely identified. Silver nanoparticles-based sensors with many nanoscale gaps with plasmonic coupling induced high local electric fields from them are, therefore, desirable and expected to exhibit high Raman signal enhancement factors and very low detection limits for low concentration or a small number of molecules. For silver nanoparticles separated by nanoscale gaps, the depth of the gaps between neighboring nanoparticles is also of nanoscale. Molecules present in the gaps are subjected to strong local electric fields and contribute to the majority of measured Raman scattering signal. This is called surface enhanced Raman scattering (SERS) effects. For a silver SERS sensor to detect very low concentration of molecules in an aqueous solution, as many as possible molecules need to be adsorbed within nanoscale gaps between silver nanoparticles and jointly contribute to the measured Raman scattering signal strength. SERS sensors have been proven to be an effective means of detecting, indentifying, and quantifying essential molecules for biomedical, environmental, and industrial applications. Besides silver, metal nanoparticles such as gold and copper are also effective in producing SERS effects. This paper reports a novel means of synthesizing an optimized density of discrete nanoscale graphene islands on copper foils by rapid thermal chemical vapor deposition in gas mixtures of hydrogen and methane. Inert gas, argon, is added as a buffer gas for diluting the combustible gas mixture of hydrogen and methane. Graphene islands on the copper foils block the chemical plating of silver nanoparticles. Only exposed copper surface which is not covered by graphene islands is allowed for the deposition of silver by chemical plating. In other words, the discrete graphene islands serve as a template for the deposition of silver nanoparticles only on exposed copper surface. These silver nanoparticles are optimized to exhibit irregular shapes compared to rounded ones formed by traditional thermal annealing of sputtering or thermal evaporation deposited silver thin films and are separated from each other by nanoscale gaps. In order to achieve high-sensitivity SERS sensors for detecting very low concentration of molecules, many research. 1536-125X © 2019 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(6) 26. IEEE TRANSACTIONS ON NANOTECHNOLOGY, Volume 19, 2020. groups have conducted extensive research on graphene synthesis and fabrication technology for optimal sizes, shapes, and distribution of silver nanoparticles. Graphene provides chemical enhancement of Raman scattering signal. However, charge transfer related chemical enhancement is much less than optimized electromagnetic enhancement, which is induced by the plasmonic coupling effects on metallic nanoparticles of suitable sizes which are separated by nanoscale gaps. In this work, graphene is used as a template to produce optimized morphology of discrete silver nanoparticles of desired sizes and shapes which are separated from each other by nanoscale gaps. Within the nanoscale gaps strong local electric fields are generated by plasmonic coupling excited by laser illumination. Irregular shaped silver nanoparticles provide additional geometric field enhancement like that between two needle-like metal tips. Graphene is a two-dimensional material that has excellent electronic, optical and thermal properties among others. A number of means of synthesizing graphene films and manufacturing graphene flakes by physical or electrochemical exfoliation of graphite have been developed. Among them, chemical vapor deposition (CVD) has emerged as the most promising technique for manufacturing graphene films for optoelectronic applications. Large-domain sizes and high-quality graphene films with a desired number of graphene layers can be routinely synthesized. Post-synthesis etching of graphene films by modern semiconductor processing techniques allows a wide variety of graphene patterns to be fabricated. However, nanoscale semiconductor fabrication processes are expensive and time consuming. Therefore, direct synthesis of nanoscale graphene structures including high-number-density nanoscale discrete graphene islands is desirable and worthy further research. In 2012, Tzeng et al. reported controlled nucleation and growth of snowflake-like graphene on copper foils by competitive growth and etching processes in hydrogen diluted methane [1]. In 2013, Wu et al. reported the growth of snowflake like graphene on molten copper with the graphene morphology being fine tuned by the ratio of applied inert gas to H2 [2]. The same group further investigated etching behaviors of monolayer and single domain graphene by varying the Ar/H2 gas flow ratio. Arrays of graphene islands of sizes smaller than 100 nm were synthesized [3]. Using a proper ratio of Ar/H2 gas flow rates, it was possible to etch a monolayer graphene film to obtain graphene patterns of six-fold symmetry [4]. Bernal-stacked bilayer graphene and rhombohedral- stacked trilayer graphene allow graphene to open its bandgap. Luo et al. prepared hierarchically stacked and patterned graphene layers by adjusting the Ar/H2 gas flow ratio and demonstrated anisotropic electrical properties of graphene [5]. In order to overcome the difficulty in applying zero-gap graphene for the fabrication of digital electronic devices, advanced lithographic processes have been resorted to fabricate graphene nanoribbons. For example, hydrogen-silsesquioxane masks were used for lateral etching of graphene by oxygen plasma to allow the narrowing of graphene nanoribbons for achieving graphene transistors with an on/off ratio of about 47 at room temperature [6]. Besides gas etching, nickel nanoparticles were also used to cut sub-10 nm graphene. nanoribbons by thermal activated movement. These efforts help increase the opportunity for graphene to become useful for future nanoscale circuits, especially those needed for digital applications [7]. Graphene films for surface-enhanced Raman scattering (SERS) applications have been studied by Tzeng et al. [8], [9] among others. In 2009, graphene was demonstrated to be a fluorescence quencher, which led to cleaner Raman signal and an improved signal-to-noise ratio [10]. Graphene has also been reported to enhance Raman scattering by chemical mechanisms based on charge transfer between the probed molecules and graphene. When polymer and thermal release tape assisted graphene transfer methods are applied, the graphene transfer process generates defects and contaminations such as carboxyl and hydroxyl groups, which help the adhesion of the probed molecules to graphene surface and lead to π-π∗ interactions and an improved charge transfer [11]. In order to further enhance SERS performance, graphene was applied along with metal nanoparticles to detect low concentration of probed molecules by means of localized surface plasmon resonance (LSPR) [12]. Hybrid graphene and plasmonic coupled metal nanoparticles were considered as a graphene-mediated SERS (G-SERS) sensor. In 2015, Leem et al. proposed a novel method to mechanically self-assemble three-dimensional crumpled graphene-gold (Au) nanoplasmonic structures, which exhibited at least one order of magnitude higher detection sensitivity than the planar graphene-Au nanoparticles system [13]. Xua et al. demonstrated a freestanding, transparent and flexible G-SERS tape for surfaces of arbitrary morphologies [14]. Zhao et al. prepared Augraphene-Ag sandwiched structures with strong electric fields in gaps separated by single layer graphene [15]. Huang et al. investigated molecular selectivity of G-SERS and found that a good enhancement factor was achieved when the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energies were in suitable positions with respect to graphene’s Fermi level. They demonstrated the G-SERS effects on molecules, with a similar energy level but different spatial structures or similar spatial structures but different energy levels [16]. In some cases, the exciting laser beam for Raman scattering brings about undesirable molecular reactions or photochemical reactions because some metal nanostructures may serve as catalysts. Therefore, an inert shell was coated on metal to isolate metal nanostructures from probed molecules [17]. By similar approaches, Xu et al. used laser molecular beam epitaxy (LMBE) to form atomically thin, inert, seamless graphene on silver nanoparticles. This substrate showed high sensitivity, high signal-to-noise ratio and good reproducibility [18]. SERS was also used to evaluate the quality of graphene and types of defects in single-layer CVD graphene [19]. Hinnemo et al. improved the graphene transfer process and led to high quality CVD graphene which was free of residues and demonstrated high SERS effects [20]. Recently, non-carbon two dimensional layered materials were used to prepare SERS substrates for investigation [21]–[23]. However, these processes require either inconvenient graphene transfer processes or expensive thin film nanofabrication technology.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(7) TZENG et al.: SILVER NANOPARTICLES SERS SENSORS USING RAPID THERMAL CVD NANOSCALE GRAPHENE ISLANDS. 27. II. EXPERIMENTAL A. Synthesis of High-Number-Density Nanoscale Discrete Graphene Islands on Copper Foils Nucleation of graphene on copper surfaces and the subsequent graphene growth to form continuous films or various graphene patterns have been extensively studied [1]. Production of largearea graphene has also become an industrial common practice. However, the synthesis of high-number-density closely spaced and discrete nanoscale graphene islands on copper instead of forming a continuous graphene film still has much room for improvement. To retain discrete graphene islands and prevent the merger of graphene islands to form larger islands or a continuous film, the growth time of graphene nuclei needs to be precisely controlled. Rapid thermal CVD provides such a capability. For reproducible results, the gas flow rate and composition need to reach a stable state before the copper foil is placed in the high temperature reaction zone for graphene nucleation and growth. The CVD nucleation and growth processes are terminated before neighboring graphene islands begin to merge. The growth time is determined based on the independently controlled flow rates of hydrogen and methane and the substrate temperature, which control the nucleation density. A desired graphene islands-based template is then obtained based on the optimal rapid thermal CVD parameters. Chemical plating time and conditions are selected to make the best use of the graphene template for the fabrication of silver nanoparticles with high concentration nanoscale gaps between silver nanoparticles. The optimal conditions for the fabrication of SERS sensors are not unique. Multiple combined sets of parameters for rapid thermal CVD and silver chemical plating may result in similarly excellent SERS sensors as long as the density, shape, and distance of nanoscale gaps are optimized. However, for economic manufacturing, easy but precise process control is required. The process time can’t be too short to be reproducible by inexpensive manipulation This paper presents one approach to achieving a very low detection limit of R6G in water by silver nanoparticles-based SERS sensors which are fabricated using graphene islands-based templates. For achieving a high graphene nucleation density on copper surface, appropriate methane flow rate and hydrogen flow rate are required. Copper serves as a catalyst for dissociation of methane and hydrogen at the synthesis temperature, at which the dissociation rate of methane and hydrogen is too low without the catalyst. Methane supplies carbon for graphene nucleation and growth. Hydrogen etches graphene, especially defects in graphene. If the ratio of methane gas flow rate to the hydrogen gas flow rate is too high, the nucleation density and the graphene growth rate are too high. The rapid thermal CVD time period is too short to be precisely controlled. Although rapid thermal CVD allows substrates to be brought into and out of the CVD reaction zone much faster than regular CVD reactors, the synthesis time should be at least one order of magnitude longer than the time it takes to load and unload the substrates. Therefore, the graphene nucleation and growth time is set to be 30 sec or longer so that the process can be precisely and reproducibly controlled by the. Fig. 1. Schematic diagram of an experimental apparatus for the rapid thermal CVD of nanoscale graphene islands on copper foils.. custom designed experimental apparatus shown in Fig. 1 which is available for this research. A schematic diagram of the graphene rapid thermal CVD apparatus is shown in Fig. 1. A mechanical vacuum pump is used to evacuate a quartz tube reactor which is placed inside a 3-zone furnace equipped with three independent electronic temperature controllers. Electronic mass flow controllers are applied to precisely control the flow rates of gases through the reaction zone. The reactor gas pressure is controlled by a throttle valve along with a capacitive manometer. A magnetically coupled sample manipulation arm allows the sample holder to be moved rapidly into and out of the high-temperature reaction zone without gas leakage. The copper foil is first moved from the high-temperature annealing zone to the low-temperature zone outside of the heated zone of the furnace. After the desired methane and hydrogen flow rates and the gas composition are stabilized, the copper foil is moved into the reaction zone again. The temperature of the copper foil rises rapidly from near room temperature to the heated furnace temperature around 1040 °C. After a preset CVD time period, the copper foil is removed from the heated furnace to a low-temperature zone outside of the furnace for cooling. Rapid thermal CVD thus precisely controls the size and concentration of graphene islands. For the synthesis of high-density discrete nanoscale graphene islands, copper foils are thermally annealed at 1040 °C for 1 hr in H2/Ar atmosphere with the H2 flow rate set at 15 sccm and Ar flow rate set at 1000 sccm. Ar is an inert gas which serves as a buffer gas for the reactor gas pressure to reach around 2 Torr but does not react with the methane, hydrogen, and copper in the graphene synthesis process. After the annealing, the copper foil is moved from the high temperature zone to a low temperature zone by a magnetically controlled arm. When the preset graphene growth conditions are established in the furnace with, for example, a steady flow of H2 (4 sccm) and CH4 (12 sccm) gas mixture while the furnace temperature is stable at 1040 °C, the copper foil is rapidly moved from the low-temperature zone back to the high-temperature zone by the same magnetically controlled arm. The graphene nucleation and growth time is varied between 30 sec and 2 min. After the nucleation and growth period, the copper foil is rapidly moved from the high temperature zone to a low temperature zone outside of the heated furnace again for cooling. Samples are characterized by Raman spectroscopy excited by a 532-nm green laser, scanning electron microscopy.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(8) 28. IEEE TRANSACTIONS ON NANOTECHNOLOGY, Volume 19, 2020. B. Electroless Chemical Plating of Silver on Exposed Copper Surface Which is Not Covered by Graphene After high-number-density discrete graphene islands are synthesized, copper foils are dipped in an aqueous solution of 5 mM AgNO3 for electroless plating of silver nanoparticles at room temperature on the exposed copper surface between neighboring graphene islands. Graphene islands serve as a mask to block the copper surface they cover from being deposited with silver from the AgNO3 aqueous solution. Besides the graphene islands displayed by the SEM images, smaller graphene nuclei are also present between them. As a result, silver nucleates around the discrete graphene islands. By varying the plating time, lateral growth time for silver nuclei is controlled and the desired size of discrete silver nanoparticles is achieved. By a precise control of the chemical plating conditions and the time period, desired silver nanostructures with high concentration of nanoscale gaps between neighboring silver nanoparticles are thus formed. Silver nanoparticles are deposited on copper surface between neighboring graphene islands and experience varied boundary conditions in different directions of lateral growth. As a result, irregular shaped instead of rounded silver nanoparticles are deposited. Irregular shapes of silver nanoparticles exhibit surfaces of large curvatures, which further promote local field. On copper surfaces without graphene islands, extensive and directional lateral growth of silver produces tree-like silver structures exhibiting multiple silver branching. For characterizing the SERS effects of copper foils coated with graphene islands and silver nanostructures, SERS sensors are dipped in aqueous solution of R6G molecules of different concentrations for 5 min. This is followed by rinsing the sensors by DI water for three times. The graphene and silver coated SERS sensors with adsorbed R6G molecules are characterized by Raman spectroscopy excited by a 532-nm green laser. III. RESULTS AND DISCUSSION A. Synthesis and Optimization of Graphene Islands-Based Templates The density and size of graphene islands synthesized on copper foils by rapid thermal CVD are determined by the hydrogen flow rate, the methane flow rate and the synthesis time period while the inert Ar gas flow rate, the temperature of copper foils and the total gas pressure are kept constant. The temperature of copper foils is set at 1040 °C. Ar gas flow rate is kept at 1000 sccm. The total gas pressure is about 2 Torr. Since the Ar gas flow rate is much higher than those of hydrogen and methane, the gas pressure only changes a little when hydrogen and methane flow rates are varied. SEM images in Fig. 2(a)–(c) show graphene islands of three different densities around 5/μm2 , 70/μm2 , and 110/μm2 synthesized for 3 min, 40 sec, and 30 sec under the hydrogen flow rate of 4, 4, and 3 sccm, the methane flow rate of 4, 12, and 15 sccm, respectively. Copper serves as a catalyst to allow the dissociation of hydrogen and methane at the synthesis temperature. Dissociated hydrogen gas etches graphene, especially defective graphene sites, at the temperature of 1040 °C. Methane gas supplies carbon for the nucleation and growth of graphene. Fig. 2. Comparison of graphene islands deposited under different methane and hydrogen flow rates and for different growth time periods on copper foils by rapid thermal CVD and corresponding Raman spectra measured after the graphene islands have been transferred to oxidized silicon substrates. (a) H2 flow rate is 4 sccm. CH4 flow rate is 4 sccm. Deposition time is 3 min; (b) H2 flow rate is 4 sccm. CH4 flow rate is 12 sccm. Deposition time is 40 sec; (c) H2 flow rate is 3 sccm. CH4 flow rate is 15 sccm. Deposition time is 30 sec. Dark areas are graphene. All scale bars are 1 µm.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(9) TZENG et al.: SILVER NANOPARTICLES SERS SENSORS USING RAPID THERMAL CVD NANOSCALE GRAPHENE ISLANDS. islands. Therefore, the higher the methane flow rate and the higher the ratio of the methane flow rate to the hydrogen flow rates, the higher the density of graphene nuclei the synthesis process will produce. With all other conditions being kept the same, the longer the synthesis time is, the larger the graphene islands will be. The graphene islands shown in Fig. 2(a) have only several graphene islands (around 5/μm2 ) in an area of one micrometer square. The nucleation density is the lowest among three displayed examples because the methane flow rate and the ratio of the methane flow rate to the hydrogen flow rate is the lowest. After graphene nuclei grow for 2 min, discrete graphene islands of about 200–300 nm in sizes are formed. The distance between areas of exposed copper surface where silver is deposited is too large for the formation of nanoscale gaps. By increasing the methane flow rate from 4 sccm to 12 sccm while keeping hydrogen flow rate the same at 4 sccm, the graphene nucleation density increases significantly. The density of graphene islands increases by more than one order of magnitude from 5/μm2 to 70/μm2 . Graphene islands remain discrete because the synthesis time is reduced to 40 sec and the size of graphene islands decreases from 200–300 to 40–80 nm. The distance between graphene islands is less than 100 nm. This is shown by the SEM image in Fig. 2(b). Graphene nucleation density can be further increased to 110/ μm2 by decreasing the hydrogen flow rate from 4 sccm to 3 sccm and increasing the methane flow rate from 12 sccm to 15 sccm while keeping other conditions the same. This is shown by the SEM image in Fig. 2(c). The size of graphene islands decreases further to 20–50 nm while the distance between graphene islands is about 50 nm. At such a high density of graphene islands, even though the synthesis time is reduced from 40 sec to 30 sec, graphene islands are shown to merge with neighboring ones. The area of exposed copper surface between neighboring graphene islands becomes even smaller than that in Fig. 2(b). This allows only a short silver deposition time before neighboring silver nanoparticles begin to merge and lose nanoscale gaps between them. The short silver deposition process is difficult to be controlled precisely and its reproducibility is poor. With less nanoscale gaps, the SERS enhancement is not as good as desired. Therefore, among these three examples, the graphene nucleation and growth shown in Fig. 2(b) is more desirable for serving as a template for subsequent deposition of silver nanoparticles. This will be further discussed later. Based on the discussion, it can be expected that the optimal size and density of graphene islands for serving as a template for the deposition of silver do not necessarily correspond to the highest density of graphene islands. The optimal size and density of graphene islands are the size and density which lead to the fabrication of the highest density of nanoscale gaps between silver nanoparticles using the graphene islands as a template. Raman scattering is applied to characterize the graphene islands shown in Fig. 2. In order to reduce the Raman background signal from the copper foil, graphene islands are transferred by the aid of polymer to SiO2 /Si substrates. A smaller graphene island has a higher ratio of the edge length to the domain area. Therefore, the Raman D peak (∼1350 cm−1 ) signal intensity increases with decreasing sizes of graphene islands. The ratio,. 29. Fig. 3. (a-c) SEM images of Ag particles plated on exposed Cu surface after the growth of graphene islands on Cu under different conditions; and (d) SEM image of tree-like Ag plated on Cu without graphene template. Graphene was grown in a mixture of 4 sccm hydrogen and 12 sccm methane at the temperature of 1040 °C. The chemical plating was carried out in 5 mM AgNO3 aqueous solution. (a) Graphene growth time is 2 min and Ag plating time is 2 min. (b) Graphene growth time is 40 sec and Ag plating time is 2 min. (c) Graphene growth time is 1 min and Ag plating time is 2 min. (d) Ag plating for two minutes on copper without graphene template.. I2D/IG, of Raman signal strengths decreases with shorter growth time and higher methane concentration. This is shown by Raman spectra shown in Fig. 2. The crystalline and electronic properties of graphene islands do not affect much on their roles as templates for chemical plating of silver nanoparticles. It should be noted that although the rapid thermal CVD time period is short compared to regular thermal CVD, graphene nucleation occurs continuous while graphene nuclei grow larger. Therefore, besides the visible graphene islands shown by SEM images, smaller and thus not clearly shown graphene islands are also present on copper surface outside the large and relatively uniform graphene islands shown in Fig. 2. These smaller graphene islands also affect the lateral growth of silver nuclei and lead the lateral profiles of silver nanoparticles to be irregular instead of being rounded silver nanoparticles which are formed by traditional thermal annealing of deposited silver thin films. Being able to deposit irregular shaped silver nanoparticles is important for SERS sensors because the higher geometric field enhancement in nanoscale gaps of irregular shaped silver nanoparticles exhibiting counter surfaces of large curvatures than that between rounded ones. B. Deposition of High-Density Silver Nanoparticles Separated by Nanoscale Gaps Fig. 3(a)–(c) show surface morphology of chemically plated silver on copper foils using graphene islands-based templates in comparison with (d) that on bare copper surface. Chemical plating of silver in silver nitrate solution occurs on exposed copper surface but not on graphene surface. Therefore, using graphene islands on copper as a template, silver nucleation and growth can be tailored to fabricate silver nanostructures exhibiting desired surface morphology. Rapid thermal CVD under similar conditions as those for synthesizing the discrete graphene islands shown in Fig. 2(b), i.e., with 4 sccm hydrogen flow and 12 sccm methane flow at the synthesis temperature of 1040 °C is applied. Different graphene islands-based templates are used.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(10) 30. IEEE TRANSACTIONS ON NANOTECHNOLOGY, Volume 19, 2020. The templates affect the surface morphology of deposited silver nanostructures, which in turn affect the plasmonic coupling effects, the strength of local electric fields, and the number density of nanoscale gaps with strong local electric fields. Silver shown in Fig. 3(a) is made by 2 min chemical plating using a graphene template synthesized by the same rapid thermal CVD conditions as that for Fig. 2(b) except for an increased synthesis time from 40 sec to 2 min. The extended graphene growth time results in larger graphene islands and in turn large silver particles. The distance between these large silver particles is too large to induce strong local electric fields by plasmonic coupling under laser illumination. The morphology is undesirable for SERS sensors. On the contrast, when the same graphene islands-based template shown by Fig. 2(b) is applied to deposit silver for 2 min, closely spaced silver nanoparticles of non-rounded shapes nearly fill up the surface of the copper foil leaving a high density of nanoscale gaps between neighboring silver nanoparticles as shown in Fig. 3(b). This is a desired morphology for achieving a high-performance silver nanoparticles-based SERS sensor. Plasmonic coupling effects will induce strong local electric fields to excite molecules which are adsorbed within nanoscale gaps between silver nanoparticles. Raman signal generated in multiple nanoscale gaps is integrated to exhibit a high enhancement factor in the measured signal from Raman scattering in molecules to be detected. Fig. 3(c) shows the morphology of silver nanoparticles deposited on a copper foil with a graphene template which is synthesized for a period of 1 min with other conditions being the same. The graphene synthesis time of 1 min is still too long resulting in merger of graphene islands and the deposition of merged silver particles. This silver morphology is not desirable for high-performance SERS sensors. Fig. 3(d) shows a tree-like silver structure deposited on a bare copper foil without a graphene template. Without graphene islands for blocking graphene nucleation and lateral growth of graphene, extensive lateral growth develops into branching of silver instead of silver nanoparticles. The silver morphology is also not desirable. In short, the silver morphologies shown in Figs. 3(a), (c), and (d) are undesirable. The silver morphology shown in Fig. 3(b) exhibits high-density of nanoscale gaps and is the desired morphology for silver nanoparticles-based SERS sensors. C. Characterization and Optimization of SERS Enhancement Factor for the Detection of Low Concentration R6G Molecules R6G molecules in aqueous solution are applied to characterize SERS sensors fabricated using graphene islands-based templates. High SERS enhancement factors and low detection limits for R6G molecules are sought after. The ratio of Raman scattering signal strength measured from adsorbed R6G molecules on SERS sensors to that on bare copper as a reference is the SERS enhancement factor. The higher the enhancement factor is, the higher the sensitivity of the SERS sensor is and the lower the detection limit it can achieve for detecting low concentration molecules. When a SERS sensor with a high enhancement factor is measured, R6G molecules of a much lower concentration are. Fig. 4. Comparison of Raman spectra measured of 10−8 M R6G adsorbed on different SERS substrates. (a) Spectra (a)-(1)-(3) are for silver nanoparticles grown for 2 min, 2 min, and 1 min, respectively, on copper foils coated with graphene nano-islands for 2 min, 40 sec, and 2 min, respectively. Spectra (a)-(4) and (5) are for tree-like silver structures grown on copper without graphene nano-islands and on a bare copper foil, repectively. On bare copper foil without graphene, R6G Raman signal can’t be observed at concentration below 10−2 M (not shown). Spectra (b)-(1) and (b)-(2) are measured from 9 × 10−12 M R6G by AgNPs/G/Cu with graphene nanoislands growth time by rapid thermal CVD and silver plating time being 40 sec and 2 min, respectively.. applied to a SERS sensor than what is applied to the reference sensor. The Raman scattering signal strengths for both cases are measured and used for the calculation of the SERS enhancement factor, which is defined by EF = (ISERS /INormal ) (CNormal /CSERS ). (1). where EF is the enhancement factor; CSERS and CNormal are concentrations of R6G molecules used for measurements by the SERS sensor and by normal Raman scattering without plasmonic coupling, respectively; ISERS and INormal are the corresponding Raman signal intensities by the same vibrational mode measured from the SERS sensor and the reference sensor, respectively. Fig. 4(a) shows Raman spectra measured from 10−8 M R6G molecules by SERS sensors. Raman spectra (1)–(3) in Fig. 4(a) are measured by silver nanoparticles deposited for 2 min, 2 min, and 1 min using graphene islands-based templates synthesized for 2 min, 40 sec, and 2 min, respectively in a gas mixture with 4 sccm hydrogen flow rate and 12 sccm methane flow rate at the synthesis temperature of 1040 °C. Spectra (4) and (5) in Fig. 4(a) are measured using tree-like silver structures deposited on bare copper and a bare copper foil without silver deposition, repectively. The detection limit for R6G molecules by the bare copper surface is 10−2 M (not shown). The enhancement factors for SERS sensors exhibiting Ramsn spectra (1) and (2) are therefore higher than 106 as calculated by dividing the detection. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(11) TZENG et al.: SILVER NANOPARTICLES SERS SENSORS USING RAPID THERMAL CVD NANOSCALE GRAPHENE ISLANDS. 31. TABLE I COMPARISON OF REPORTED SERS DETECTION LIMITS FOR R6G. Fig. 5. Raman spectra of (1) 10−10 M and (2) 10−16 M R6G measured by AgNPs/G/Cu SERS sensors with graphene nanoislands growth time by rapid thermal CVD and silver plating time being 40 sec and 2 min, respectively.. limit of the copper reference at 10−2 M by the 10−8 M R6G molecules which produce clear Raman scattering signal. Fig. 4(b) shows Raman spectra measured from 9 × 10−12 M R6G molecules adsorbed on silver nanoparticles chemically plated for 2 min using graphene islands-based templates made by rapid thermal CVD for 40 sec and 2 min, respectively. The graphene template is shown in Fig. 2(b) and the corresponding SERS sensor shown Fig. 3(b) are used for the measurement of the Raman spectrum shown in Fig. 4(b)-(1). The SERS sensor shown in Fig. 3(a) is used to measure the Raman spectrum shown in Fig. 4(b)-(2). The SERS sensor shown in Fig. 3(b) produces much higher Raman signal strength than that shown in Fig. 3(a), which use graphene templates synthesized for a longer period of time. By comparing the Raman signal strength shown in Fig. 4(b)-(1) measured from 9 × 10−12 M R6G molecules with the reference detection limit of 10−2 M R6G molecules on a bare copper surface, the enhancement factor is calculated to be higher than 109 . SERS sensors are optimized by selecting the most suitable chemically plating time for depositing discrete silver nanoparticles which are separated from each other by nanoscale gaps. Graphene islands-based templates are optimized by selecting the most suitable hydrogen and methane flow rates for graphene nucleation and selecting the growth time of the graphene nuclei to form discrete graphene islands. Both optimization processes are carried out by repetitive cycles of characterizing the surface morphology by SEM and fine tuning of the process parameters. Detection of R6G molecules of ultra-low concentration between 10−12 and 10−14 M has also been reported by other research groups [18], [25]–[27], [29], [31]. For the detection of R6G molecules at a very low concentration of 10−12 to 10−16 M, it is important for R6G molecules to be adsorbed in the nanoscale gaps instead on other areas where local electric fields are weak. Fig. 5 shows that plasmonic coupling based SERS sensors made of silver nanoparticles can detect very low concentration of R6G molecules at as low as 10−16 M. Characteristic Raman peaks of R6G molecules at 611 and 773 cm−1 are displayed. By comparing the signal intensities of the 611 cm−1 peak with that. measured from 10−2 M R6G molecules adsorbed on a bare Cu foil, the enhancement factor is on the order of 1014 . At such a low concentration of R6G, the measured Raman signal strength is weak but still readable. The irregular shaped silver nanoparticles deposited by chemical plating under the restrictions by graphene islands further contribute to the very low detection limit of R6G. For the same amount of electric charges of opposite signs on two counter surfaces of large curvatures across a nanoscale gap, the geometric field enhancement effect induces an even higher local electric fields than that between two planar surfaces or two surfaces of small curvatures. With a large concentration of nanoscale gaps between silver nanoparticles and high field enhancement due to irregular (non-rounded) shapes of silver nanoparticles, Raman scattering signals originated from adsorbed R6G molecules in many nanoscale gaps add up and the characteristic Raman peaks can be detected. A comparison between the SERS enhancement factor achieved in this work with selected data in the literature is shown in Table I. The list is by no means exhaustic. Some SERS sensors on this list exhibit high enhancement factor due to three-dimensional nanostructures which possess large effective surface areas for adsorbing R6G molecules. The total signal strength therefore increases with the effective surface. SERS sensors reported in this paper has a planar array of silver nanoparticles optimized to have a very high density of nanoscale gaps where plasmonic coupling induced strong local electric fields and SERS enhancement occur. Enhancement factors are based on the selected reference sensor and are thus relative values. In this paper, the detection limit of a bare copper foil is used as a reference for comparisons. All SERS sensors capable of detecting R6G molecules of 10−12 M or lower are excellent. Nevertheless, it is certain that the unique silver-graphene-copper SERS molecular sensors reported in this paper can be easily and inexpensively manufactured. They are capable of detecting and identifying very low concentration of R6G molecules. They perform as well as or even better. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(12) 32. IEEE TRANSACTIONS ON NANOTECHNOLOGY, Volume 19, 2020. than reported SERS sensors which rely on three-dimensional nanostructures [29] made of silver and other suitable metal nanoparticles. D. Graphene Islands as Protective Coatings and Chemical Enhancer Besides graphene islands-based templates for the fabrication of excellent silver SERS sensors by chemical plating, additional roles of graphene islands are worthy discussion. Since silver nanoparticles are selectively deposited on copper foils where no graphene islands are present, silver nanoparticles are sourrounded by graphene islands. Some graphene islands are partially covered by silver nanoparticles due to lateral growth of silver nanoparticles. Therefore, graphene islands also serve as protective coatings for copper surface where silver nanoparticles are not present. It thus prevents the copper surface from oxidation and other reactions by the environments a SERS sensor is subjected to. Graphene is known to be chemically stable and an excellent diffusion barrier for most materials including oxygen. Heating in the ambient air at 200 °C for 10 min doesn’t cause significant adverse effects on graphene islands. Without protection by graphene islands, exposed copper surface may be oxidized, oxidation of copper under silver nanoparticles by oxygen diffusion may cause silver nanoparticles to lose adhesion to the SERS sensor. Oxidized copper may introduce additional background fluorescence signals to the measured Raman spectra resulting in reduced Raman signal to noise ratio. Therefore, graphene islands are expected to be beneficial to long-term stability of the SERS sensors. Graphene provides charge-transfer related chemical enhancement to Raman scattering signal strength. Although the optimal chemical enhancement alone is weak compared to well demonstrated high electromagnetic enhancement factors, chemical enhancement does add to the overall SERS enhancement. It is difficult to precisely separate the contributions made by chemical enhancement and electromagnetic enhancement when both play roles to some extents. Nevertheless, an estimation of what graphene may contribute to the overall enhancement is worthwhile. For a continuous graphene film synthesized on copper surface, the detection limit for R6G molecules has been measured to be 10−6 M. This corresponds to a SERS enhancement factor of about 104 in reference to a bare copper surface. Raman enhancement of continuous graphene is mainly attributed to chemical enhancement related to charge transfer processes. For discrete graphene islands, the enhancement factor of nanoscale graphene islands has been measured to be as high as 105 , which is about 10 times higher than that of a continuous graphene film. Part of the enhancement is attributed to the nanoscale gap (30∼50 nm) between neighboring graphene islands and possibly to the nanoscale roughness of graphene resulting from mismatched coefficients of thermal expansion between graphene (α-graphene = −6 × 10−6 /K at 27 °C) and copper (α−Cu = 24 − 10−6 /K). This is only a qualitative estimation of possible contributions by graphene on the overall enhancement factors. In the presence of both silver nanoparticles. and graphene islands, potential roles of graphene may be different from graphene alone. SERS enhancement factors also depend on whether molecules are preferrably adsorbed in nanoscale gaps. A SERS sensor capable of detecting ultra low concentration of molecules such as R6G does not guarantee the same high sensitivity for other molecules. Charge carrying molecules such as adenine is an example. Effects of different probe molecules on SERS enhancement factors will be reported and discussed in a separate paper. IV. CONCLUSION High density, discrete and nanoscale graphene islands with optimized size and distribution for serving as templates for chemical plating of silver nanoparticles for high-sensitivity SERS sensors have been synthesized by rapid thermal CVD. The optimized graphene islands-based templates result in electroless plating of irregularly shaped and discrete silver nanoparticles on copper surface exhibiting high density and uniformly distributed nanoscale gaps between silver nanoparticles. Irregularly shaped silver nanoparticles provide further field enhancement between surfaces of silver nanoparticles with large curvatures. Charge transfer based chemical enhancement by graphene contributes minor SERS enhancement. Graphene templates provide protection of copper from undesired reactions with the environments which the SERS sensors are subjected to. Optimized SERS sensors fabricated by this method exhibit enhancement factors as high as 1014 . This innovative and useful application of graphene islands as templates for the fabrication of unique and excellent SERS sensors with ultra-high SERS enhancement factors and ultra-low detection limits for R6G molecules are expected to find additional applications beyond SERS sensors. This invention has been awarded a Taiwan, ROC patent I632354 [32] and a US patent 10,429,308 [33]. REFERENCES [1] Y. Tzeng, K. Liang, C. Y. Liu, C. C. Chang, and Y. Wu, “Controlled nucleation and growth of graphene: Competitive growth and etching in hydrogen diluted methane,” Proc. 12th IEEE Nanotechnol. Conf., Birmingham, U.K., Aug. 2012, pp. 20–23. [2] B. Wu et al., “Self-organized graphene crystal patterns,” NPG Asia Mater., vol. 5, 2013, Art. no. e36. [3] D. Geng et al., “Direct top-down fabrication of large-area graphene arrays by an in situ etching method,” Adv. Mater., vol. 27, pp. 4195–4199, 2015. [4] D. Geng et al., “Fractal etching of graphene,” J. Amer. Chem. 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(13) TZENG et al.: SILVER NANOPARTICLES SERS SENSORS USING RAPID THERMAL CVD NANOSCALE GRAPHENE ISLANDS. [10] L. Xie, X. Ling, Y. Fang, J. Zhang, and Z. Liu, “Graphene as a substrate to suppress fluorescence in resonance raman spectroscopy,” J. Amer. Chem. Soc., vol. 131, pp. 9890–9891, 2009. [11] J.-C. Yoon, P. Thiyagarajan, H.-J. Ahn, and J.-H. Jang, “A case study: Effect of defects in CVD-grown graphene on graphene enhanced Raman spectroscopy,” RSC Adv., vol. 5, pp. 62772–62777, 2015. [12] Y. Zhao et al., “Plasmonic-enhanced Raman scattering of graphene on growth substrates and its application in SERS,” Nanoscale, vol. 6, 2014, Art. no. 13754 [13] J. Leem, M. C. Wang, P. Kang, and S. W. Nam, “Mechanically selfassembled, three-dimensional graphene−gold hybrid nanostructures for advanced nanoplasmonic sensors,” Nano Lett., vol. 15, pp. 7684–7690, 2015. [14] W. Xua et al., “Surface enhanced Raman spectroscopy on a flat graphene surface,” Proc. Nat. Acad. Sci., vol. 109, no. 24, pp. 9281–9286, 2012. [15] Y. Zhao, W. Zeng, Z. Tao, P. Xiong, Y. Qu, and Y. Zhu, “Highly sensitive surface-enhanced Raman scattering based on multi-dimensional plasmonic coupling in Au–graphene–Ag hybrids,” Chem. Commun., vol. 51, pp. 866–869, 2015. [16] S. Huang et al., “Dresselhaus, molecular selectivity of Graphene-enhanced raman scattering,” Nano Lett., vol. 15, pp. 2892–2901, 2015. [17] J. F. Li et al., “Shell-isolated nanoparticle-enhanced Raman spectroscopy,” Nature, vol. 464, pp. 392–395, Mar. 2010. [18] S. Xu et al., “High performance SERS active substrates fabricated by directly growing graphene on Ag nanoparticles,” RSC Adv.„ vol. 5, 2015, Art. no. 90457 [19] D. L. Matz, H. Sojoudi, S. Graham, and J. E. Pemberton, “Signature vibrational bands for defects in CVD single-layer graphene by surface-enhanced Raman spectroscopy,” J. Phys. Chem. Lett., vol. 6, pp. 964–969, 2015. [20] M. Hinnemo et al., “Scalable residue-free graphene for surface-enhanced Raman scattering,” Carbon, vol. 98, pp. 567–571, 2016. [21] C. Muehlethaler, C. R. Considine, V. Menon, W.-C. Lin, Y.-H. Lee, and J. R. Lombardi, “Ultrahigh Raman enhancement on monolayer MoS2 ,” ACS Photon., vol. 3, pp. 1164–1169, 2016. [22] L. Sun et al., “Plasma modified MoS 2 nanoflakes for surface enhanced Raman scattering,” Small, vol. 10, no. 6, pp. 1090–1095, 2014.. 33. [23] Xi Ling et al., “Raman enhancement effect on two-dimensional layered materials: Graphene, h-BN and MoS2 ,” Nano Lett., vol. 14, pp. 3033– 3040, 2014. [24] Q. Cai et al., “Catalytic degradation of dye molecules and in situ SERS monitoring by peroxidase-like Au/CuS composite,” Nanoscale, vol. 6, no. 14, pp. 8117–8123, 2014. [25] B. Daglar, “Anemone-like nanostructures for non-lithographic, reproducible, large-area, and ultra-sensitive SERS substrates,” Nanoscale, vol. 6, no. 21, pp. 12710–12717, 2014. [26] Y. Li et al., “A facile fabrication of large-scale reduced graphene oxide– silver nanoparticle hybrid film as a highly active surface-enhanced Raman scattering substrate,” J. Mater. Chem. C, vol. 3, no. 16, pp. 4126–4133, 2015. [27] J. Guo et al., “Graphene oxide-Ag nanoparticles-pyramidal silicon hybrid system for homogeneous, long-term stable and sensitive SERS activity,” Appl. Surf. Sci., vol. 396, pp. 1130–1137, 2017. [28] S. Xu, S. Jiang, J. Wang, J. Wei, W. Yue, and Y. C. Ma, “Graphene isolated Au nanoparticle arrays with high reproducibility for highperformance surface-enhanced Raman scattering,” Sensors Actuators B, Chem., vol. 222, pp. 1175–1183, 2016. [29] Y. Zhao et al., “Toward highly sensitive surface-enhanced Raman scattering: The design of a 3D hybrid system with monolayer graphene sandwiched between silver nanohole arrays and gold nanoparticles,” Nanoscale, vol. 9, no. 3, pp. 1087–1096, 2017. [30] H.-X. Mei et al., Tuning SERS properties of pattern-based MWNTs-AuNP substrates by adjustment of the pattern spacings,” Carbon, vol. 136, pp. 38– 45, 2018. [31] F. Huang, G. Ma, J. Liu, J. Lin, X. Wang, and L. Guo, “High-Yield synthesis of hollow octahedral silver nanocages with controllable pack density and their High-Performance sers application,” Small, vol. 12, no. 39, pp. 5442– 5448, 2016. [32] Y. Tzeng and Y. R. Chen, “Raman spectroscopy and method of manufacturing the same,” ROC Patent I632354, Aug. 11, 2018. [33] Y. Tzeng and Y. Chen, “Carrier of Raman Spectroscopy and method of manufacturing the same,” US Patent #10,429,308, Oct. 1, 2019.. Authorized licensed use limited to: National Cheng Kung Univ.. Downloaded on October 28,2020 at 06:01:51 UTC from IEEE Xplore. Restrictions apply..

(14) biosensors Article. Silver SERS Adenine Sensors with a Very Low Detection Limit Yonhua Tzeng *. and Bo-Yi Lin. Department of Electrical Engineering, National Cheng Kung University, Tainan 701, Taiwan; [email protected] * Correspondence: [email protected] . Received: 5 April 2020; Accepted: 13 May 2020; Published: 15 May 2020. Abstract: The detection of adenine molecules at very low concentrations is important for biological and medical research and applications. This paper reports a silver-based surface-enhanced Raman scattering (SERS) sensor with a very low detection limit for adenine molecules. Clusters of closely packed silver nanoparticles on surfaces of discrete ball-like copper bumps partially covered with graphene are deposited by immersion in silver nitrate. These clusters of silver nanoparticles exhibit abundant nanogaps between nanoparticles, where plasmonic coupling induces very high local electromagnetic fields. Silver nanoparticles growing perpendicularly on ball-like copper bumps exhibit surfaces of large curvature, where electromagnetic field enhancement is high. Between discrete ball-like copper bumps, the local electromagnetic field is low. Silver is not deposited on the low-field surface area. Adenine molecules interact with silver by both electrostatic and functional groups and exhibit low surface diffusivity on silver surface. Adenine molecules are less likely to adsorb on low-field sensor surface without silver. Therefore, adenine molecules have a high probability of adsorbing on silver surface of high local electric fields and contribute to the measured Raman scattering signal strength. We demonstrated SERS sensors made of clusters of silver nanoparticles deposited on discrete ball-like copper bumps with very a low detection limit for detecting adenine water solution of a concentration as low as 10−11 M. Keywords: SERS; silver; adenine; R6G; graphene; plasmon; Raman scattering; copper. 1. Introduction Adenine is one of aromatic bases found in DNA and RNA. It is soluble in water only at a low concentration. Shown in Figure 1 is a schematic diagram of the chemical structure of an adenine molecule. Adenine adsorption on silver and gold nanoparticles and electrodes exhibits desirable surface-enhanced Raman scattering (SERS) signal strength, which is suitable for the detection of diseases, DNA hybridization and many biomedical and agricultural applications [1]. Therefore, it has become one of the most frequently studied biomolecules along with the rapid increase in SERS research in recent years [2–4]. Adenine molecules adsorb on metal surfaces by strong and complex interactions. The interactions include electrostatic forces and coupling by functional groups. In most reported cases, adenine molecules adsorb on silver metal at a tilted angle to the surface [2]. Some planar adsorption was also reported [5]. The strong interactions with silver metal surface lead to the low diffusivity of adenine molecules on silver surface. The characteristics result in different SERS detection limits for adenine molecules from that for Rhodamine 6G molecules [5–11]. Recently, we reported a novel means of applying nanoscale graphene islands on copper foils as a template or a mask to selectively chemically plate silver nanoparticles on the surface of a copper foil without graphene coverage [12,13]. A two-dimensional array of discrete but closely spaced silver nanoparticles was deposited on a planar copper foil using a graphene. Biosensors 2020, 10, 53; doi:10.3390/bios10050053. www.mdpi.com/journal/biosensors.

(15) Biosensors 2020, 10, 53. 2 of 13. template. By gradually increasing the silver deposition time and repetitively measuring the distance between silver nanoparticles, high-density nanoscale silver gaps were optimized. Graphene islands, which serve as a mask to block silver deposition by chemical plating, remain on the copper surface and are surrounded by silver nanoparticles. A low SERS detection limit of R6G molecules well below 10−12 M was achieved routinely. By optimizing the gap spacing and the density of nanoscale gaps, 10−16 M R6G molecules was detected. This extremely low detection limit by a SERS sensor was found to be not applicable to adenine molecules. The detection limit of adenine molecules by the same SERS sensor was found to be many orders of magnitude higher than that of R6G molecules. For example, a detection limit on the order of 10−6 M adenine molecules was measured in comparison with that of 10−12 –10−16 M for R6G molecules. Aiming at finding an effective means of improving the detection limit for adenine molecules, this study was carried out to resolve the difference in sensitivity of SERS sensors to R6G Biosensors 2020, 10, x FOR PEER REVIEW 2 of 14 molecules from adenine molecules.. Figure 1. Chemical structure of an adenine molecule.. Figure 1. Chemical structure of an adenine molecule.. For a SERS sensor to detect a specific kind of molecules at a very low concentration, it is necessary to increase the probability of the molecules to adsorb on sensor, where local electric fields are very Adenine molecules adsorb on metal surfaces by strong and complex interactions. The interactions high so that the enhanced Raman scattering signal strength is high. Plasmonic coupling has been include electrostatic forces and coupling by functional groups. In most reported cases, adenine found to be an effective means of inducing very high local electric fields [14,15]. If a molecule adsorbs molecules adsorb on silver metal at a tilted angle to the surface [2]. Some planar adsorption was also in a low-field area, the molecule contributes little to the measured Raman scattering signal strength. reported [5]. The strong interactions with silver metal surface lead to the low diffusivity of adenine The low detection limit that is being pursued is adversely affected. On the other hand, it is necessary molecules on silver surface. The characteristics result in different SERS detection limits for adenine for the sensor to have as many as possible “hot spots” where plasmon-induced local electric fields molecules from that for Rhodamine 6G molecules [5–11]. Recently, we reported a novel means of are very high. The main mechanisms for molecules to adsorb on a SERS sensor vary for different applying nanoscale graphene islands on copper foils as a template or a mask to selectively chemically molecules. For achieving a low detection limit for different molecules, different SERS design strategies plate silver nanoparticles on the surface of a copper foil without graphene coverage [12,13]. A twoare therefore required. The best SERS sensor for adenine molecules may be different from that for dimensional array of discrete but closely spaced silver nanoparticles was deposited on a planar copper R6G molecules. foil using a graphene template. Byon gradually the the silver deposition time and repetitively The adsorption of molecules a sensor increasing surface where local electromagnetic field is weak measuring the distance between silver nanoparticles, high-density nanoscale silver gaps were should be minimized. Instead, adsorption of molecules to “hot spots” where high local electric fields optimized. Graphene islands, which serve as a mask to block silver deposition by chemical plating, are induced by plasmonic coupling should be promoted. For molecules which interact with and adsorb remain on easily, the copper surrounded by silver nanoparticles. A lowmost SERSmolecules detectionwill limit on silver there surface should and be noare silver in the low-field areas. By these means, −12 M was achieved routinely. By optimizing the gap spacing and the ofadsorb R6G molecules well below 10 on a high-field sensor surface and contribute to the measured Raman scattering signal strength. density gaps, 10−16are Mwasted R6G molecules was detected. This extremely detection limit by As fewof asnanoscale possible molecules due to adsorption in low-field areas of low the sensor. a SERSThe sensor was found to be not applicable to adenine molecules. The detection adenine detection limit of adenine molecules by gold and silver-based SERS sensors haslimit beenof reported −8 −9 molecules by the from same10 SERS found to be many orders of magnitude higher thanof that of R6G to range mainly Msensor to 10 was M [16–20]. Some research groups reported the detection adenine −6 M adenine molecules was measured molecules. a detection limit on the order of10 10−11 moleculesFor at aexample, lower concentration of 10−10 M [21] and M [22]. In order to achieve a 10−11 Min −16 M for R6G molecules. Aiming at finding an effective means of comparison withsemiconductor that of 10−12–10 detection limit, nanofabrication technology was applied [22]. improving the detection limit of forinteracting adenine molecules, carried to resolve the difference R6G molecules, instead favorablythis withstudy silverwas surfaces of out positive potential, interact inwith sensitivity of SERS sensors to R6G molecules from adenine molecules. graphene with a negative potential. When a graphene mask is present below nanogaps formed For a silver SERS nanoparticles, sensor to detect specific kind molecules a veryon low it is between R6Ga molecules are of more likely toatdiffuse theconcentration, surface of silver nanoparticles to enterthe nanogaps where enhancement factor is the strongest. R6G molecules necessary to increase probability of the the SERS molecules to adsorb on sensor, where local electric fields are very high so that the enhanced Raman scattering signal strength is high. Plasmonic coupling has been found to be an effective means of inducing very high local electric fields [14,15]. If a molecule adsorbs in a low-field area, the molecule contributes little to the measured Raman scattering signal strength. The low detection limit that is being pursued is adversely affected. On the other hand, it is necessary for the sensor to have as many as possible “hot spots” where plasmon-induced local electric.

(16) Biosensors 2020, 10, 53. 3 of 13. accumulate in the nanogaps, where the local electromagnetic field induced by plasmonic coupling is very strong and the included Raman scattering signal strength is significantly enhanced. R6G molecules are more likely than adenine molecule to adsorb inside abundant silver nanogaps in an array of close spaced silver nanoparticles deposited on a planar copper surface. This is believed to have resulted in much lower detection limit for R6G molecules than adenine molecules. 2. Materials and Methods The process of fabricating a two-dimensional array of silver nanoparticles on a planar copper foil as illustrated in Figure 2A has been described in reference [12,13]. Figure 2B,C show the deposition of clusters of silver nanoparticles on discrete copper structures. The copper structure is formed after the copper thin film on the silicon surface melts at high temperature during annealing and the graphene thermal CVD processes. Copper films are deposited by RF magnetron sputtering in Ar gas using 60 W RF power at 13.56 MHz for 15-min pre-sputter cleaning. It is followed by applying RF power of 90 W for sputter coating of copper films of a desired thickness by controlling the sputtering time. Copper films which are thinner than 100 nm peel off easily from the silicon surface under the copper annealing and graphene growth conditions at 900 ◦ C. Broken, peeling off, and distorted pieces from 100 nm thick copper films scatter around a planar silicon surface. This is illustrated by Figure 2B. Distribution of copper pieces on a planar silicon surface is random. They are not suitable for the fabrication of a reproducible SERS sensor. In order to collect molten copper in pre-arranged location for achieving uniformly distributed copper structures for subsequent chemical plating of silver nanoparticles, holes are chemically etched into a planar silicon wafer [23]. A sputtered copper film of thinner than 100 nm melts, flows and solidifies inside etched holes. Subsequent rapid thermal CVD of graphene on the ball-like copper bumps creates a template for the selective deposition of silver nanoparticles only on copper surfaces where there are no graphene islands to block the chemical plating of silver. Silver grows vertically from ball-like copper bumps surface to form a cluster of elongated nanoparticles pointing in different directions as illustrated by Figure 2C. Tables 1 and 2 show the remaining detailed process parameters for hole etching, thermal annealing of copper thin films, and rapid thermal CVD of graphene islands on copper. Silver nitrate solution was prepared by adding 100 mL deionized water to 0.08493 g of silver nitrate powder (Sigma-Aldrich, Inc., purity >99.8%, St. Louis, MO, USA) at room temperature followed by thorough stirring. SERS samples were immersed in 3 mL silver nitrate solution for a pre-determined period of time and then removed for blowing dry by nitrogen. Chemical plating of silver on copper surface, where there is no graphene deposition was applied to deposit silver nanoparticles. In the chemical plating of silver on copper, copper metal dissolves in the silver nitrate solution. Silver deposits on the copper surface [24]. Silver does not deposit on graphene surface. The balanced equation for the reaction is Cu(s) + 2AgNO3 (aq)——->Cu(NO3 )2 (aq) + 2Ag (1) According to Product Number A8626, CAS #: 73-24-5, Sigma-Aldrich, Inc., adenine is soluble at 1 part to ~2000 parts cold water. In order to ensure full dissolution, adenine (Sigma, purity >99%) of 0.5 mg was dissolved in deionized water to make 3.7 × 10−6 M adenine in water solution. The solubility of adenine in water is low and depends on temperature and other conditions. The initial concentration of adenine in water solution at 3.7 × 10−6 M is well below the solubility of adenine in water at room temperature. This adenine solution was further diluted by deionized water by 10 times each time and repetitively to prepare a series of adenine water solution of different concentration. The order of magnitude of the concentrations, i.e., 10−6 , 10−7 , 10−8 , 10−9 , 10−10 , 10−11 , 10−12 M were used for evaluation of SERS sensors..

(17) Biosensors 2020, 10, 53. 4 of 13. After the SERS sensor was immersed in adenine water solution of a known concentration for 10 min, the sensor was removed from the solution and blown dry by nitrogen. The Horiba Scientific Raman system with a green laser at 532 nm and a laser power at 450 mW was used to measure Raman spectra. The Raman system has an optical fiber optical system with a 100 times object lens. The laser focus was optimized to measure the highest Raman scattering signal of the test sample. Diamond crystals with a Raman peak at 1332 cm−1 are often used for calibration. The laser beam was focused on the sensor surface in an area of about 10 µm in size. Raman spectra were measured in 5–10 different areas on each sensor for each concentration of adenine water solution. The concentration of adenine is considered to be above the low detection limit if the Raman scattering peak at 760 cm−1 , which is characteristic of adenine molecules, is clearly distinguishable from the background signal. The low detection limit was determined by the lowest concentration of adenine solution, which produced a clearly detectable Raman scattering peak. Multiple sensors were characterized. 4 of 14 Biosensors 2020,adenine 10, x FOR PEER REVIEW. Figure 2. Schematic diagram of silver nanoparticles grown by nitrate reduction on graphene nano-. Figure 2. Schematic diagram of silver nanoparticles grown by nitrate reduction on graphene nano-islands islands template covered copper surfaces. (A) Two-dimensional array of silver nanoparticles templatedeposited coveredoncopper surfaces. (A) Two-dimensional array of silver nanoparticles deposited on a a planar copper surface using graphene nano-islands synthesized on copper by rapid planar copper surface using nano-islands synthesized onthe copper by rapid thermal CV technique thermal CV technique graphene as a template. The red sections represent graphene template. (B) Thermal as a template. The sections represent template. (B) Thermal annealing of a copper annealing of ared copper thin film depositedthe on graphene a planar silicon wafer breaks apart the copper thin films into discrete pieces, which peel off wafer from the siliconapart surface, or deform into discrete three- pieces, thin film deposited on a planar silicon breaks themelt copper thin films into discrete dimensional structures. Graphene nano-islands deposited onthree-dimensional the copper structurescopper serve asstructures. a which peel off fromcopper the silicon surface, melt or deform into discrete template for silver nanoparticles to grow by nitrate reduction in directions perpendicular to the Graphene nano-islands deposited on the copper structures serve as a template for silver nanoparticles to copper surfaces. (C) A copper thin film is deposited on a silicon wafer with etched holes. Broken grow bycopper nitrate reduction in directions perpendicular to the copper surfaces. (C) A copper thin film is thin films melt and solidify at the bottom and on the sidewall of etched holes. Discrete silver deposited on a silicon withon etched Broken thin films melt and solidify at the bottom nanoparticles arewafer deposited copperholes. surfaces using copper graphene nano-islands synthesized on copper and on the sidewall of etched holes. Discrete silver nanoparticles are deposited on copper surfaces using surfaces as a template. graphene nano-islands synthesized on copper surfaces as a template. Table 1. Process Parameters for Etching Holes in Silicon Wafers.. Step 1 Etching SiO2 layer H2O2 10 mL HF 10 mL Immersion 1 h. Step 2 Etching Si layer CuSO4 40 mM H2O2 10 mL H2O 50 mL HF 13 mL etching 30 min. Step 3 Clean with DI water DI water.