Multi-Color Enhanced Fluorescence Imaging of a Breast Cancer Cell with A Hole-Arrayed Plasmonic Chip.

Breast cancer cells of MDA-MB-231 express various types of membrane proteins in the cell membrane. In this study, two types of membrane proteins in MDA-MB-231 cells were observed using a plasmonic chip with an epifluorescence microscope. The targeted membrane proteins were epithelial cell adhesion molecules (EpCAMs) and epidermal growth factor receptor (EGFR), and Alexa®488-EGFR antibody and allophycocyanin (APC)-labeled EpCAM antibody were applied to the fluorescent detection. The plasmonic chip used in this study is composed of a two-dimensional hole-array structure, which is expected to enhance the fluorescence at different resonance wavelengths due to two kinds of grating pitches in a square side and a diagonal direction. As a result of multi-color imaging, the enhancement factor of Alexa®488-EGFR and APC-EpCAM was 13 ± 2 and 12 ± 2 times greater on the plasmonic chip, respectively. The excited wavelength or emission wavelength of each fluorescent agent is due to consistency with plasmon resonance wavelength in the hole-arrayed chip. The multi-color fluorescence images of breast cancer cells were improved by the hole-arrayed plasmonic chip.

Study on the Mechanism of Diarylethene Crystal Growth by In Situ Microscopy and the Crystal Growth Controlled by an Aluminum Plasmonic Chip.

The microcrystalline film of an open-ring isomer (1o) of diarylethene 1 was prepared on an Al plasmonic chip with a grating structure. Photoisomerization from 1o to the closed-ring isomer (1c) and growth of needle-shaped crystals in 1c were observed in situ under an upright-inverted microscope. In the center part of the film, crystal growth of needle-shaped-crystal of 1c was observed upon UV irradiation from the top side, but not upon UV irradiation from the bottom side. However, crystallization occurred at the edge of the film upon UV irradiation from the bottom side. It was suggested that crystal growth of 1c required a high mobility of 1c near the film surface. Furthermore, the existence of 1o platform is also found to be required for alignment of 1c molecules by the results under the irradiation from the bottom and top sides. With the Al plasmonic chip, the conversion rate from 1o to 1c was larger inside the grating by the plasmonic enhanced field. Therefore, when the attenuated UV light was irradiated to the film edge with high mobility of 1c from the bottom side, the conversion rate was more than 60%, and the needle-shaped crystals of 1c were observed only inside the grating area. Crystal growth was controlled by the conversion rate of 1c promoted inside the grating. From the above, the larger conversion rate of 1c more than 60%, a high mobility of 1c near the film surface or edge, and the existence of the 1o platform for alignment of 1c molecules, are considered to be required for crystal growth in 1c.

Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull's Eye-Type Plasmonic Chip.

Surface plasmon field-enhanced fluorescence microscopic observation of a live breast cancer cell was performed with a plasmonic chip. Two cell lines, MDA-MB-231 and Michigan Cancer Foundation-7 (MCF-7), were selected as breast cancer cells, with two kinds of membrane protein, epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR), observed in both cells. The membrane proteins are surface markers used to differentiate and classify breast cancer cells. EGFR and EpCAM were detected with Alexa Fluor® 488-labeled anti-EGFR antibody (488-EGFR) and allophycocyanin (APC)-labeled anti-EpCAM antibody (APC-EpCAM), respectively. In MDA-MB231 cells, three-fold plus or minus one and seven-fold plus or minus two brighter fluorescence of 488-EGFR were observed on the 480-nm pitch and the 400-nm pitch compared with that on a glass slide. Results show the 400-nm pitch is useful. Dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed with seven-fold plus or minus two and nine-fold plus or minus three, respectively, on the 400-nm pitch pattern of a plasmonic chip. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelengths. An optimal grating pitch of a plasmonic chip improved a fluorescence image of membrane proteins with the help of the surface plasmon-enhanced field.

Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells Using Multicolor Fluorescence Microscopy with a Plasmonic Chip.

A plasmonic chip was applied to 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 anti-EpCAM 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 fluorescence images of APC-EpCAM to be obtained on the plasmonic chip that were more than 10 times brighter 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 the cell membrane in contact with 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 membranes contacting the chip.

Polydopamine Thin Films as Protein Linker Layer for Sensitive Detection of Interleukin-6 by Surface Plasmon Enhanced Fluorescence Spectroscopy.

Polydopamine (PDA) thin films are introduced to the surface modification of biosensor surfaces utilizing surface plasmon enhanced fluorescence spectroscopy (SPFS) as the linker layer of capture antibody on to the sensor surfaces. The capture antibody can be directly attached to the sensor surface without using any coupling agent by functionalizing the gold sensor surface with PDA thin films. The PDA coating is performed by a single-step preparation process by applying the dopamine solution on the sensor surface, which requires an extremely short incubation time (10 min). The real-time in situ measurement of the adsorption kinetics of the capture antibody onto the PDA-coated sensor surface is studied by surface plasmon resonance (SPR) spectroscopy. It reveals that the immobilization of capture antibody immediately occurs after introduction of a solution containing capture antibody, and the sensor surface is fully covered with the capture antibody. The sensitive detection of the cytokine marker interleukin-6 (IL-6) is performed by SPFS using a sandwich assay format with fluorescently labeled detection antibody. The sensor chips functionalized by PDA chemistry exhibited sensitive sensor responses with low nonspecific adsorption of the detection antibody onto the sensor surface. The detection limit of IL-6 with the developed SPFS biosensor is determined to be 2 pg/mL (100 fM), which is within the range of the diagnostic criteria. Our observation elucidates the remarkable utility of PDA coatings for chemical modification of the metallic sensor surfaces by a simple, brief, and inexpensive manner.

Sensitive detection of a tumor marker, α-fetoprotein, with a sandwich assay on a plasmonic chip.

Two types of plasmonic silver- and gold-coated grating biosensor chips (plasmonic chip) were applied in the detection of α-fetoprotein (AFP) with a sandwich imunoassay and surface plasmon field-enhanced fluorescence. On the plasmonic chip, unlabeled marker in the sandwich immunoassay was first quantitatively detected over a wide range between 10(-12) and 10(-8) g/mL. The affinity constants between AFP and anti-AFP antibody, which were obtained by fitting the experimental data to the Langmuir isotherm adsorption curve, were 1 × 10(8) g(-1) mL regardless of the kind of metal in the plasmonic chips. Although the fluorescence intensity on the silver plasmonic chip was 5 times larger than that on the gold plasmonic chip, the limit of detection (LOD) was on the order of 10(-11) g/mL and not improved with a silver plasmonic chip. Herein, we used a new setup that generated less dispersions of both the fluorescence intensity for nonspecific adsorption and the background (optical blank) signal and improved the LOD of AFP to 4 pg/mL (55 fM) with the silver plasmonic chip. With the highly sensitive detection in the sandwich immunoassay, the development of a plasmonic chip for clinical diagnosis by a blood test is promising.

Application of 300× enhanced fluorescence on a plasmonic chip modified with a bispecific antibody to a sensitive immunosensor.

The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300× enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface.