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Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells by Using Multicolor Fluorescence Microscopy with a Plasmonic Chip Keiko Tawa, Shohei Yamamura, Chisato Sasakawa, Izumi Shibata, and Masatoshi Kataoka ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07777 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016
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ACS Applied Materials & Interfaces
Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells by Using Multicolor Fluorescence Microscopy with a Plasmonic Chip
Keiko Tawa†,§ *, Shohei Yamamura¶, Chisato Sasakawa†,§, Izumi Shibata¶, Masatoshi Kataoka¶
†
Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
§
AIST, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8377, Japan
¶
AIST, 2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan
Phone: +81-79-565-9758, E-mail:
[email protected] ACS Paragon Plus Environment
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ABSTRACT: Plasmonic chip was applied to the live cancer cell imaging. The epithelial cell adhesion molecule (EpCAM) is a surface marker that can be used to classify breast cancer cell lines into distinct differentiation states. EpCAM and the nuclei of two kinds of living breast cancer cells, MDA-MB231 and MCF-7, were stained with allophycocyanin (APC)-labeled antiEpCAM antibody and 4′,6-diamidino-2-phenylindole (DAPI), respectively, and the cells were scattered on either a plasmonic chip (metal-coated wavelength-scale grating substrate) or a control glass slide. Multicolor fluorescence microscopic imaging allowed more than 10 times brighter fluorescence images of APC-EpCAM to be obtained on the plasmonic chip compared with those on the glass slide. In contrast, in the fluorescence images of DAPI-stained nuclei, no difference in brightness was observed between substrates. The fluorescence enhancement of APC-EpCAM in cell membrane contacting to the plasmonic chip is thought to be due to the excitation of APC molecules localized within the surface plasmon field. Analysis of the cross section of a fluorescence image revealed a distribution of EpCAM at a higher level of fluorescence in the center of the cell image because of contact between the cell membrane and the plasmonic chip. In contrast, fluorescence images of APC-EpCAM taken on a glass slide were so dark that only the outline of the cell was characterized. The plasmonic chip thus constitutes a simple and powerful tool for analyzing the distribution and kinetics of surface marker proteins in cell membrane contacting to the chip.
KEYWORDS: multicolor fluorescence imaging, microscopy, breast cancer cells, living cell, plasmon, grating
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ACS Applied Materials & Interfaces
Introduction Recently, the application of plasmonics, i.e., scientific technology and academic studies relating to the theory of surface plasmon resonance (SPR), to immunosensing and bioimaging has developed rapidly, and numerous papers in this field have been published.1–5 Various nanoparticles and nanostructures have been fabricated by using instruments that are typically used for the manufacture of semiconductors, and the optical characteristics and localized plasmonic features of these systems have been shown.6–8 Propagated plasmonic features have also been studied with prism-coupled SPR using Kretschmann configuration and with gratingcoupled (GC)-SPR.9,10 The fabrication process required to construct propagated-SPR systems is not so complex, and mass production is easier to achieve compared with localized-SPR systems. Furthermore, propagated-SPR can provide a homogeneous, enhanced field over a wide area, which offers a significant advantage for applications in biological research. As one of the applications of propagated-SPR, the plasmonic chip (a metal-coated substrate with grating structure; Figure 1) provides enhanced fluorescence because of the enhanced electric field that is brought about by direct coupling of incident light on the metal surface. Direct coupling without the use of special optics such as a prism is very convenient for microscopic observation.10,11 When a plasmonic chip is placed on a sample stage instead of a cover glass, a bright fluorescence image can be obtained as a result of GC-surface plasmon enhanced fluorescence.12–14 Plasmonic chips thus have great potential for biological applications. In our previous studies, 10-fold brighter fluorescence images of neuron cells cultivated on a plasmonic chip15 were recorded with an epi-fluorescence microscope, and the target protein in an immunoassay was detected until 4-order lower concentration16–18 on a plasmonic chip.
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The observation of living cells and the application of multicolor imaging are important for numerous medical fields. In this study, the distribution of epithelial cell adhesion molecule (EpCAM), which is a surface marker that is used to differentiate and classify breast cancer cells, has been studied in two types of living breast cancer cell lines,19–21 Michigan Cancer Foundation7 (MCF-7) and MDA-MB 231. The images were obtained with an epi-fluorescence microscope by using either a control glass slide or a plasmonic chip. The cells were multicolor stained with 4′,6-diamidino-2-phenylindole (DAPI), which stained the cell nucleus, and allophycocyanin (APC)-labeled anti-EpCAM antibody, which stained EpCAM molecules contained in the cell membrane, and then the cells were dispersed onto the plates.19–21 Generally, the level of expression of EpCAM in MDA-MB 231 is known to be lower than that in MCF-7, and imaging of the former with APC stain by using conventional glass slides is difficult.19 For early diagnosis and detection of cancer cells, sensitive fluorescence imaging techniques are required that allow the detection of very low levels of EpCAM. In this study, fluorescence images of APC-EpCAM in cancer cell lines were clearly recorded by using a plasmonic chip, and the distribution of EpCAM within the cell on the chip was determined.
EXPERIMENTAL METHODS Fabrication of a Plasmonic Chip. A periodic replica was fabricated with PAK-02A (Toyo Gosei, Tokyo, Japan) dropped on the cover glass by using the UV-nanoimprint method. The grating structure was as shown in Figure 1, and the pitch and hole depth were 480 nm and 30 nm, respectively. The grating was covered with Ti, Ag, Ti, and SiO2 thin films of thickness
