Highly Reproducible Au-Decorated ZnO Nanorod Array on a Graphite

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Highly reproducible Au-decorated ZnO nanorod array on a graphite sensor for classification of human aqueous humors Wansun Kim, Soo Hyun Lee, Sang Hun Kim, Jae-Chul Lee, Sang Woong Moon, Jae Su Yu, and Samjin Choi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16130 • Publication Date (Web): 03 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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ACS Applied Materials & Interfaces

Highly reproducible Au-decorated ZnO nanorod array on a graphite sensor for classification of human aqueous humors

Wansun Kima,1, Soo Hyun Leeb,1, Sang Hun Kimb, Jae-Chul Leec, Sang Woong Moond*, Jae Su Yub*, Samjin Choia,c*

a. Department of Medical Engineering, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea b. Department of Electronics and Radio Engineering, Kyung Hee University, Gyeonggi-do 17104, Republic of Korea c. Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea d. Department of Ophthalmology, College of Medicine, Kyung Hee University, Seoul 05278, Republic of Korea

*Address for correspondence: Sang Woong Moon, M.D. & Ph.D., Jae Su Yu, Ph.D., Samjin Choi, Ph.D. Department of Biomedical Engineering, College of Medicine, Kyung Hee University 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, South Korea Tel: +82 2 961 0290; fax: +82 2 961 5515 E-mail addresses: [email protected] (S.W. Moon), [email protected] (J.S. Yu), [email protected] (S. Choi)

1

These authors contributed equally to this work.

*Keywords: ZnO nanorod; graphite sheet; surface-enhanced Raman scattering (SERS); finite element method (FEM) computation; aqueous humor

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Abstract Gold-decorated, vertically grown ZnO nanorods (NRs) on a flexible graphite sheet (Au/ZnONRs/G) were developed for surface-enhanced Raman scattering (SERS)-based biosensing to identify trace amounts of human aqueous humors. This Au/ZnONRs/G SERS-functionalized sensor was fabricated via two steps: hydrothermal synthesis-induced growth of ZnO NRs on graphite sheets for nanostructure fabrication, followed by e-beam evaporatorinduced gold metallization on ZnONRs/G for SERS functionalization. The thickness of the Au layer and the height of the ZnO NRs for enhancing SERS performance were adjusted to maximize Raman intensity, and the optimized Au/ZnONRs/G nanostructures were verified by the electric finite element computational models to maximize the electric fields. The proposed Au/ZnONRs/G SERS sensor showed an enhancement factor of 2.3×106 via rhodamine 6G Raman probe and excellent reproducibility (relative standard deviation of 98.5%) was prepared by Daejung Chemical & Metals (Siheung, Korea). All chemicals were of reagent grade and used without any further purification.

2.2. Preparation of aqueous humors 2.2.1. Selection of patient groups Human aqueous humors were collected at Kyung Hee University Hospital at Gangdong in Seoul, Republic of Korea. All human aqueous humor biofluids were obtained during surgical procedures. Informed consent was obtained from 15 patients (72±11 years), who included five cataract patients, five AMD patients and five DME patients. All procedures involving humans adhered to the Declaration of Helsinki and were approved by the Kyung Hee University Medical Center guidelines (IRB 2015-07-42).

2.2.2. Collection of aqueous humors Each aqueous humor was taken from a patient about to undergo surgery for cataracts (control group) or intravitreal bevacizumab injection (AMD and DME groups). A 0.1-mL volume of pure aqueous humor was aspirated using a 30-gauge needle attached to a tuberculin microsyringe through limbal paracentesis. The collected aqueous humor samples were placed immediately into sterile tubes and stored at –80°C in a deep freezer until further analysis.32

2.3. Preparation of Au/ZnONRs/G The Au/ZnONRs/G was fabricated by the following procedures (Scheme 2). A graphite sheet with a size of 2×2 cm2 was cleaned in ethanol and then dried with nitrogen (N2) gas. A ZnO seed layer was deposited on the graphite sheet using a radiofrequency (RF) magnetron sputtering system (KVS-3004, Korea Vacuum Tech., Ltd., Gimpo, Korea). The sputtering was carried out at a working pressure of 0.80 Pa (6 mTorr) with 100 W RF power in an Ar environment. Then, 25 mM Zn(NO3)2·6H2O and 25 mM HMTA were dissolved in 200 mL of de-ionized (DI) water at room temperature, and the resulting solution was continuously stirred with a magnetic bar for 2 hr for homogeneity. ZnO NRs were vertically grown on the surface of the ZnO seed-coated-graphite sheet in a thermal oven at 90°C. The height of the ZnO NRs was controlled by varying the growth time. They were rinsed with droplets of DI water and dried under an N2 flow. The Au layer was deposited on the ZnONRs/G using an e-beam evaporator (KVE-E2004, Korea Vacuum Tech., Ltd.), with a constant ratio of 1 Å/s at a high voltage of 7 kV under a vacuum of 2.7 mPa (0.02 mTorr).

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Scheme 2. Fabrication procedure for Au/ZnONRs/G. (A) Preparation of an ethanol-cleaned and N2 gas-dried 2×2 cm2 graphite sheet. (B) Deposition of ZnO seed layer on the as-prepared graphite sheet by sputtering. (C) Growth of ZnO NRs on the graphite sheet via the hydrothermal synthesis based on zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and hexamethylenetetramine (HMTA). (D) Au deposition on ZnONRs/G by the e-beam evaporation.

2.4. Ophthalmological evaluations All patients underwent ophthalmic examinations at baseline, including best-corrected visual acuity, fundus examination with slit-lamp biomicroscopy and spectral-domain optical coherence tomography (OCT). Fundus photographs of all patients were obtained using Visucam (Carl Zeiss Meditec, Jena, Germany). Spectral-domain OCT (Heidelberg Engineering, Heidelberg, Germany) examinations were performed with the macular area (6×6 mm2) centered on the fovea. High speed mode imaging was performed with automated real-time features and nine frames.

2.5. Characterization of Au/ZnONRs/G The morphologies and electron analysis of Au/ZnONRs/G were characterized using a field-emission scanning electron microscope (FE-SEM; LEO SUPRA 55, Carl Zeiss, Jena, Germany) with an accelerating voltage of 10 kV and a scanning transmission electron microscope (STEM; JEM-2100F, JEOL, Tokyo, Japan) with an accelerating voltage of 200 kV. For the TEM analysis, the Au/ZnONRs/G was pretreated by a focused ion beam (FIB; Quanta 3D FEG, FEI Company, Eindhoven, Netherlands) milling with 0.1 nA at 30 kV. The crystalline phases of Au/ZnONRs/G were characterized by X-ray diffractometer (XRD; D8 Advance, Bruker, Madison, WI, USA) with Cu Kα X-rays in the 2θ range of 30 to 90° with a step of 0.02°. Raman spectra were measured using a SENTERRA confocal Raman spectroscope (Bruker Optics, Billerica, MA, USA) and a 785-nm diode laser with 10 mW power and a 20× objective lens with a numerical aperture of 0.4. All Raman spectra were obtained within the fingerprint range of 417–1782 cm–1 with a spectral resolution of 5 cm–1 and twice the acquisition time of 10 s, at six random points. To evaluate the large-scale uniformity of the Au/ZnONRs/G, 400-point Raman spectra were collected in a 100×100 µm2 area with a pitch of 5 µm via an integration time of 1 s. A 1µL analytic droplet of the sample was used to analyze the Raman spectrum.

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2.6. Computational electrodynamics The SERS effect of Au/ZnONRs/G on the electromagnetic (EM) fields was simulated with commercial COMSOL Multiphysics software (COMSOL Inc., Burlington, MA, USA). The 2D finite element method (FEM) models were constructed according to our statistical findings for fabrication condition-dependent Au/ZnONRs/G sensors. The near-field distribution of the electric fields was calculated to solve Maxwell equations with given boundary conditions (BCs). Illumination came from the Au layer side with polarization along the dimer axis, and the input source electric field was E0=1 V/m. The excited wavelength was 785 nm.

2.7. Multivariate statistical analysis The principal component analysis (PCA) algorithm used to extract the global spectral features of the three types of aqueous humors was implemented with MATLAB software (MathWorks Inc., Natick, MA, USA) as the multivariable analytical tool. All spectral vectors normalized by Z-scores were used as the transfer function to determine the principal component (PC) scores for each aqueous humor spectrum. A simple mapping method was used to project two global parameters selected manually among the three PC scores acquired for a 2D space. Multi-support vector machine (SVM) classifiers with a one-against-one approach onto the 2D plot were used to classify the cataract, AMD and DME diseases after leave-one-out cross-validation.

3. Results and discussion 3.1. Morphology of Au/ZnONRs/G Figure 1 shows the FE-SEM images of the ZnONRs/G and Au/ZnONRs/G. The bare graphite substrate had a rough surface and multi-layer characteristics (Fig. S1). The structure of the ZnO NRs strongly depended on the solution concentration and deposition time.34 The solution concentration, which is proportional to the diffusion of Zn2+ ions to nuclei, affects the diameter of the ZnO NRs, and ZnO bulk film-like structures are formed at high solution concentrations. A 25 mM Zn(NO3)2·6H2O aqueous solution enabled the growth of the vertically aligned ZnO NRs with an average diameter of 56 nm, which were densely grown over a wide area of substrate (Fig. 1A and B).35 Furthermore, the hydrothermal synthesis time determines the height of the ZnO NRs. Four different synthesis times (40, 70, 120 and 160 min) led to the growth of ZnO NRs with heights of 100, 200, 400 and 600 nm, respectively. We preliminarily compared the SERS performance between two gold deposition techniques, e-beam evaporation and sputtering (Fig. S2). The gold was deposited with a thickness of 100 nm on three different ZnONRs/G sensors. The e-beam evaporation yielded gold lumps at the heads of the ZnONRs/G due to poor step coverage, while the sputtering resulted in gold

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conformal coatings (Fig. S3). The e-beam-evaporated sample revealed 10-fold higher SERS activity than the sputtered one, because e-beam evaporation led to the formation of a compact gold film compared to the rough gold grains created by the sputtering. This finding was consistent with the silver metallization technique by e-beam evaporation, which yielded higher Raman signals compared to silver coating by sputtering.36 Thus, e-beam evaporation was chosen as the default gold deposition method for further experiments.

Figure 1. FE-SEM images of the ZnONRs/G and Au/ZnONRs/G at (A) 150× and (B) 200k× magnification. (C) Variations in Raman intensity with different Au thicknesses on ZnO NRs with heights of 100, 200, 400 and 600 nm. * indicates the optimal fabrication conditions. Tilted FE-SEM images of 200-nm-thick Au-decorated 400-nm-high ZnO NRs on graphite sheets at (D) 200k×, (E) 50k× and (F) 5k× magnification.

Raman intensities were optimized by adjusting the thickness of the Au layer and the height of the ZnO NRs (Fig. 1C). Since most commercial Au SERS substrates had a thickness of 200 nm, and the 600-nm-height ZnO NR substrate showed a lower SERS intensity than the 200-nm-high ZnO NR substrate (Fig. S2), Au layers with thicknesses between 0 and 200 nm and ZnO NRs with heights between 100 nm and 600 nm were evaluated. The Raman peak at 1361 cm–1 was used as a representative R6G molecule-characterized peak (Table S1). Overall, Raman intensities increased as Au thickness increased for a given height of ZnO NRs.37 A 200-nm-thick Au layer on ZnO NRs with a height of 400 nm showed the highest R6G-induced Raman intensities and a reproducibility of

Highly Reproducible Au-Decorated ZnO Nanorod Array on a Graphite

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