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Science of Advanced Materials Vol. 6, pp. 2491–2495, 2014 (www.aspbs.com/sam)

Silver Nanostar Patterned Substrate for Label-Free Characterization of Breast Cancer Cells based on Surface-Enhanced Raman Spectroscopy Md. Khaled Hossain1 , Hyeon-Yeol Cho2 , Kyeong-Jun Kim2 , and Jeong-Woo Choi1, 2, ∗ 1

Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-Ro, Mapo-Gu, Seoul, 121-742, Republic of Korea 2 Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-Ro, Mapo-Gu, Seoul, 121-742, Republic of Korea

ABSTRACT

KEYWORDS: Silver Nanostar, Label-Free Characterization, Breast Cancer Cells, SERS.

1. INTRODUCTION The characterization of cancer cells is important for personalized anticancer therapy.1 Among many techniques, immunofluorescence-based techniques are more common for characterization and sub-typing of cancer cells.21 3 SERS nanotags can also be used for characterization and subtyping,4 but in both cases additional labeling of cells is required, which involves steps that are complex and time-consuming. Furthermore, fluorescent or Raman dyes may produce cytotoxicity and fluorescent dyes have a photobleaching effect.5 Hence, label-free detection methods are very important. Previously, label-free detection of normal and cancer cells was conducted electrochemically,6 and single cells in droplets electrically.7 Although electrochemical signals can distinguish between normal and cancer cells, subtyping of cells of the same origin is not possible with electrochemical methods. In the case of electrical detection, a microfluidic chip, which may be costly, is required. For this reason, there is still a need for ∗

Author to whom correspondence should be addressed. Email: [email protected] Received: 9 July 2014 Accepted: 20 October 2014

Sci. Adv. Mater. 2014, Vol. 6, No. 11

non-destructive methods for label-free detection of cells. Raman spectroscopy is a powerful analytical technique that enables rapid, reagent-free, and non-destructive analysis of living cells.8 The biochemical composition of cells can be thoroughly studied by peak-by-peak analysis of Raman spectra.9 However, the application of Raman spectroscopy to cell-based analyses is very limited due to its weak and unstable signal.10 Surface-enhanced Raman spectroscopy (SERS) leverages certain phenomena to offer an exciting opportunity to overcome the critical disadvantages of normal Raman spectroscopy. SERS can be achieved by using noble metal nanoparticles either in colloidal form or in deposited form on a solid substrate.11 However, the use of colloidal nanoparticles for SERS creates problems due to aggregation, and therefore using a substrate modified with a metal nanostructure pattern is a better option for measuring SERS. Previously a lot of surface modification method has been reported.121 13 However, the fabrication of SERSactive substrates has been found to have a number of problems, including poor signal enhancement, uniformity, reproducibility, or requiring further processing for removal of the template.14 Hence, an advanced method for the

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doi:10.1166/sam.2014.2227

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Characterization of cancer cells is important in case of personalized cancer therapy. Cells can be characterized based on their surface marker expression level using fluorescence or surface-enhanced Raman spectroscopy (SERS) method, but in both cases its needed additional labeling with fluorescent or Raman dyes, those may cause cellular cytotoxicity. In this study, we report silver (Ag) nanostar pattern modified ITO substrate capable of label free characterization of breast cancer cells based on SERS. The substrate was fabricated by depositing homogeneously distributed silver nanostar pattern on ITO-glass surface electrochemically. The substrate was capable to produce highly intense SERS spectra in comparison to gold nanopattern modified substrate. Two different subtypes of breast cancer cells (SK-BR-3 and MCF-7) were immobilized on the substrate separately Delivered by Publishing Technology to: Ji Young Lee and successfully distinguishedIP:based on the molecular information detected by SERS. Our newly developed 163.239.37.72 On: Wed, 21 Jan 2015 06:57:54 substrate can be used as an effective platform for molecular detection and characterization of different cells Copyright: American Scientific Publishers originated from same or different organs.

Silver Nanostar Patterned Substrate for Label-Free Characterization of Breast Cancer Cells based on SERS

fabrication of an SERS-active surface is required for more effective enhancement of Raman signals. In this paper, we report a simple, one-step, and templatefree method for the fabrication of a highly sensitive Ag nanostar modified ITO substrate and its application to the analysis of the biochemical composition of different breast cancer cell lines, as well as their characterization and subtyping. Ag nanostar array modified ITO substrates were fabricated by the electrochemical deposition of Ag from AgNO3 solution in the presence of polyethylene glycol (PEG) as a surfactant on an ITO surface (Fig. 1).

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2. EXPERIMENTAL DETAILS

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at 25 ž C in an electric-heated thermostatic water bath. In order to remove any trace of surfactants that may have been adsorbed on the Ag nanostar modified ITO surface, the substrates were rinsed in DIW and boiled for 5 min with isopropyl alcohol. The active area for electrochemical deposition of Ag nanostars was 20 mm × 10 mm. Nanostar surface morphologies were analyzed by a field effect scanning electron microscope (FE-SEM) JSM-6700F. The surface plasmon resonance of the substrate was measured using a Jasco V-530 UV/VIS spectrometer. A cell culture chamber unit with the dimensions 1 cm × 1 cm × 1 cm (width × length × height) was attached to the Ag nanostar/ITO surface using polydimethylsiloxane (PDMS).

2.1. Materials 2.3. Immobilization of RGD Peptide on the Substrate Silver nitrate (AgNO3 ), polyethylene glycol (PEG), phosA solution of 0.01 mg/mL of RGD (Arg-Gly-Asp) peptide phate buffered saline (PBS), and 4-amino thiophenol was prepared in DI water, added on the chip, and main(4-ATP) were purchased from Sigma-Aldrich (St. Louis, tained at 4 ž C for 12 hours. Finally, the peptide modified MO, USA). RGD peptide was obtained from R&D Syselectrode was exposed to a sterilized environment for cell tems (Minneapolis, MN, USA). RPMI media, fetal bovine seeding. serum (FBS), antibiotics (penicillin and streptomycin), and trypsin (0.05% trypsin, 0.53 mM EDTA-4Na) were 2.4. Cell Culture purchased from Gibco (Invitrogen, Grand Island, USA). Breast cancer cell lines (MCF-7, SK-BR-3) were obtained Deionized water (DI), obtained from a millipore water sysfrom ATCC (Manassas, VA, USA). The cells were cultured tem was used throughout the experiment. All other chemat 37 ž C in RPMI1640 medium supplemented with 10% icals were analytical grade reagents. Delivered by Publishing Technology to: Ji Young heat-inactivated FBS Lee and 1% antibiotics (penicillin and IP: 163.239.37.72 On: Wed,streptomycin) 21 Jan 2015in06:57:54 a humidified atmosphere of 95% air with 2.2. Fabrication of Silver Nanostar Copyright: Pattern onAmerican Scientific Publishers 5% CO2 . The cells were grown in tissue culture-grade petri ITO (ITO/Ag) Substrate dishes. At 80% confluency, the cells were sub-cultured at ITO-coated glass substrates were cleaned by sonication for a density of 1 × 105 cells/mL on culture plates, and then 15 min using 1% Triton X-100 solution, deionized water incubated for 3–4 days. For SERS experiments, the cells (DIW) and ethanol sequentially, followed by basic piranha were transferred into the chip at a known cell density by solution (1:1:5, H2 O2 :NH3 :H2 O) for 30 min at 80 ž C. infusion of new culture medium. Finally, the substrates were cleaned by DIW, and then dried under a N2 stream to obtain a clean ITO surface. Ag 2.5. Raman Spectroscopy nanostar arrays were electrochemically deposited on ITO 48 hours after incubation, the biochemical composition substrates (20 mm×20 mm) using a 1.0 mM AgNO3 aqueous solution containing PEG (17 mg/mL) as a surfactant. of living MCF-7 and SK-BR-3 cells was investigated The potential was maintained at −0.9 V (vs. Ag/AgCl) and by Raman spectroscopy using Raman NTEGRA spectra the deposition temperature was controlled and maintained (NT-MDT, Russia). Before measuring SERS spectra from the cell, a SERS map of the cell was made by selecting a 35 Œm × 35 Œm area with 32 × 32 data points. Raman spectra were recorded using a near-infrared (NIR) laser emitting light at a wavelength of 785 nm. Ten scans of 1 s from 500 cm−1 to 1700 cm−1 were recorded, and the means of these scans were used to construct a curve.

3. RESULTS AND DISCUSSION

Fig. 1. Illustration for substrate preparation, immobilization of cells, and detection of SERS spectra.

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3.1. Fabrication of Silver Nanostar Patterned Substrate and Its Plasmon Absorption Ag nanostar arrays were fabricated by the deposition of Ag from a AgNO3 aqueous solution in the presence of PEG as a surfactant using an electrochemical deposition method (Fig. 2). Figure 2(a) shows a topographic SEM image of the Ag nanostar array fabricated on the ITO surface with Sci. Adv. Mater., 6, 2491–2495, 2014

Hossain et al.

Silver Nanostar Patterned Substrate for Label-Free Characterization of Breast Cancer Cells based on SERS

a uniform distribution of nanostars. Figure 2(c) demonstrates the UV-Vis spectra of the ITO/Ag substrate. The substrate exhibited two surface plasmon absorption bands: A strong band at 439 nm likely produced from the spherical nanostructures, and a broad band at 613 nm likely produced from the nanostars.15 Since the enhancement of surface electric field depends on surface plasmon excitation, Ag nanostars may strongly absorb energy and scatter the electromagnetic field. Hence, the Ag nanostar modified ITO substrate should lead to a high enhancement in the Raman signals. 3.2. Study of SERS Properties of the Ag Nanostar Modified ITO Substrate To study the SERS properties of the substrate, a Raman reporter, 4-ATP (6 ŒM), was immobilized on the substrate and SERS spectra were measured using a 785 nm NIR laser. Before measuring SERS, a SERS map was made from the 4-ATP immobilized substrate (Fig. 2(b)). The SERS map shows a homogenous distribution of hotspots. The SERS spectra of the 4-ATP molecule were measured with a 1 s exposure time. The Raman bands at 1079, 1414, and 1575 cm−1 are due to “CS, 7a(a1 ), „CH+“CC, 3(b2 ), and “CC 8b (b2 5 mode, respectively.16 The SERS results demonstrated that the 4-ATP layer on the ITO/Ag Sci. Adv. Mater., 6, 2491–2495, 2014

substrate produced a significant increase in the intensity of the overall spectra. We also compared the SERS intensity between the Ag nanostar modified and Au nanopattern modified ITO (ITO/Au) substrates. The results show that the overall SERS intensity of ATP (Fig. 2(g)) of the ITO/Ag substrate is approximately 2.2 times higher than that of the ITO/Au substrate. 3.3. SERS Spectra of Living Breast Cancer Cells The SERS mechanism depends on two major enhancement factors: an electromagnetic enhancement called the electric effect, and a chemical enhancement called the charge-transfer effect. The electromagnetic enhancement has been found to play a key role in the SERS effect.17 The Ag nanostar structures allow for ready excitation of plasmons on the Ag nanostars’ surfaces, which localizes and increases the concentration of the electric field density. Due to the difference in the dielectric constants between the Ag nanostar surface and the surrounding media, the electromagnetic energy density of the Ag nanostars could be a source of the electromagnetic enhancement that is a major contributor to SERS efficacy. Moreover, Ag nanostars have a high value of surface roughness due to their structural characteristics, and induce electric field changes that can radiate both parallel and perpendicular to the 2493

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Fig. 2. Confirmation of topology and SERS propertyby of Publishing novel metal modified ITO substrate; silver (a)–(c),Lee gold (d)–(f). SEM image of (a) ITO/Ag and Delivered Technology to: Ji Young (d) ITO/Au substrate; inset shows high magnification image of a nanostar. SERS map image2015 of 4-ATP immobilized (b) ITO/Ag and (e) ITO/Au substrate. IP: 163.239.37.72 On: Wed, 21 Jan 06:57:54 UV-vis spectra of the (c) ITO/Ag and (f) ITO/Au substrate. (g) SERS spectra of the 4-ATP immobilized on ITO/Ag and ITO/Au. (h) Comparison of Copyright: American Scientific Publishers SERS intensity of both substrates. Scale bar (a) 10 Œm, (d) 500 nm.

Silver Nanostar Patterned Substrate for Label-Free Characterization of Breast Cancer Cells based on SERS

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Table I. Assignment of SERS spectra measured from MCF-7 and SKBR-3 cells. Bands (cm−1 )

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645 780 820 852 883 936 1030 1060 1157 1230 1340 1377 1437 1471 1490

Tentative assignments

Molecular origin

Cell type

Tyr DNA Tyr Tyr Trp C–C backbone Phe C–C C–C, C–N str. Amide-III Trp DNA (T) CH2 C–H def./bending DNA (G, A)

Proteins Nucleic acids Proteins Prpteins Proteins Lipids Proteins Lipids Proteins Proteins Proteins Nucleic acids Lipids Proteins Nucleic acids

MCF-7, SK-BR-3 SK-BR-3 MCF-7 SK-BR-3 MCF-7 MCF-7 MCF-7 SK-BR-3 MCF-7, SK-BR-3 MCF-7, SK-BR-3 MCF-7 SK-BR-3 MCF-7 MCF-7 SK-BR-3

spectra with a 785 nm NIR laser within the spectral range from 500 cm−1 to 1700 cm−1 . The SERS spectra of the Fig. 3. Characterization of MCF-7 by SERS. (a) Bright field, (b) SERS living cells consist of a series of bands corresponding map image and (c) Raman spectra of a MCF-7. Bare ITO substrate (dash), to all biopolymers found in cells (Figs. 3 and 4). The ITO/Ag substrate (solid). tentative assignment of the measured SERS spectra from MCF-7 and SK-BR-3 cells are presented in Table I.141 18–20 surface. Thus, whenever an incident photon falls on the Figures 3to:and 4 showLee the differences in the contents of Delivered by Publishing Technology Ji Young roughened surface, the excitationIP: of the plasmon resonance biomolecules in the two 163.239.37.72 On: Wed, 21 Jan 2015 06:57:54different breast cancer cells studof the metal can occur to allow scattering. ied. ThePublishers MCF-7 cells had high phenylalanine, tyrosine, Copyright: American Scientific The distribution of biopolymers in living MCF-7 and and tryptophan content (Raman bands at 1030, 1060, and SK-BR-3 breast cancer cells was studied using the SERS 852 cm−1 respectively). The experimental results indicate that the Ag nanostar modified substrate can be used successfully to obtain molecular information from cells through SERS. Therefore, using the Ag nanostar modified ITO substrate, it is easy to determine the biochemical composition of cells and distinguish them based on their chemical contents. Hence, our newly developed technique can be used as an effective method for the characterization and sub-typing of cells with high efficiency and reproducibility.

4. CONCLUSION

Fig. 4. Characterization of SK-BR-3 by SERS. (a) Bright field, (b) SERS map image and (c) Raman spectra of a SK-BR-3. Bare ITO substrate (dash), ITO/Ag substrate (solid).

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In this study, we introduced a simple method for the characterization of cancer cells using Ag nanostar modified ITO substrates. The Ag nanostar modified ITO surface enabled the sensitive detection of the biochemical composition of living cells without cellular damage in a short detection time. Compared with a Au nanopattern modified ITO substrate, Raman signals from the Ag nanostar modified ITO substrate showed significantly increased signals from 4-ATP, confirming its superior ability to enhance optical signals from analytes. Our study’s development of the SERS technique using a Ag nanostar modified ITO substrate can be readily applied for sensitive biochemical characterization and distinguishing of cells originating from the same or different organs. Furthermore, it has Sci. Adv. Mater., 6, 2491–2495, 2014

Hossain et al.

Silver Nanostar Patterned Substrate for Label-Free Characterization of Breast Cancer Cells based on SERS

potential applications for characterization of cancer for personalized therapy and monitoring of chemotherapeutic effects of anti-cancer drugs. Acknowledgments: The research was supported by Leading Foreign Research Institute Recruitment Program through the National Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP) (2013KIA4A3055268) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 2014R1A2A1A10051725).

References and Notes

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