Enhanced fluorescence microscopy with the Bull's eye-plasmonic chip.

A Bull's eye-plasmonic chip composed of concentric circles was applied to enhanced fluorescence microscopy. Among one dimensional (1-D), 2-D, and Bull's eye periodic structures, the Bull's eye-plasmonic chip provided the most enhanced fluorescence intensity under the epi-fluorescence microscope, because incident light through the objective lens with all azimuthal angles can be effectively applied to the surface plasmon resonance- field (excitation field) and the plasmon-enhanced emission was also effectively collected. In the fluorescence observation of a single nanoparticle, the enhanced fluorescence images for a microsphere with ϕ 2 µm and a nanosphere with ϕ 200 nm were observed. For the nanospheres with ϕ 40 and 20 nm, the fluorescence image, which was undetectable on a glass slide, was observed in a spatial resolution of roughly diffraction limit on the Bull's eye-plasmonic chip. Furthermore, the use of an appropriate pinhole at the aperture stop in the incident optical system improved the fluorescence enhancement. The applicability of a Bull's eye-plasmonic chip to fluorescence imaging was demonstrated.

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.

Properties of modified surface for biosensing interface.

Properties of modified surface, behavior against salting-out effect, suppressive effect for protein nonspecific adsorption, and wettability were examined using various mercapto compounds bearing methyloligoethylene glycol, oligoethylene glycol, alkyl oligoethylene glycol, alkyl phosphoryl choline, alkyl inverse phosphoryl choline, and alkyl sulfobetaine moieties. The behavior against salting-out effect was examined using gold nanoparticle with PBS and NaCl aqueous solution. The suppressive effect for protein nonspecific adsorption was evaluated by SPR, and the wettability was measured on the SPR chip. The gold nanoparticle modified with 8C3EG, 12C4EG, 12CPC, 6CCP, and 12CCP showed excellent behavior against salting-out effect. The suppression of protein nonspecific adsorption was effective with 6EG, 12C4EG, 12CPC, and 12CS. On the other hand, the modified surface possessed high wettability except for the surface modified with M6EG. The results indicate that incorporation of alkyl group into surface modification materials is effective for the enhancement of behavior against salting-out effect and suppressive effect for protein nonspecific adsorption regardless of wettability. Among the zwitter ionic derivatives, inverse phosphoryl choline derivatives showed intriguing properties, high behavior against salting-out effect with high wettability but low suppressive effect for protein nonspecific adsorption.

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.