High spatial resolution surface plasmon resonance imaging using a plasmonic chip.

The surface plasmon resonance (SPR) technique has been widely applied to biosensing technologies for the rapid quantification of biomolecules without enzyme and fluorescent labeling. However, the conventional prism-coupling SPR method generally has a detection area of a few mm2, and the large contribution of the background signal forms a barrier to highly sensitive detection. Based on a highly spatially resolved SPR method, the present study constructed a scanning GC-SPR imaging instrument using an objective lens with a high numerical aperture and a plasmonic chip that could be used for grating-coupled SPR. Focusing light on the diffraction limit can suppress background signals and improve detection sensitivity. SPR imaging can also be performed by scanning a focal spot. Using this method, the refractive index of a mixture of water and dimethyl sulfoxide was measured with a detection accuracy of 2.43 × 10-3 RIU. Polydopamine films prepared with a thickness of <5 nm were also measured, and each film thickness was evaluated with high sensitivity from the effective refractive index detected in a small area of <1 µm2.

Nanoantenna effect dependent on the center structure of Bull's eye-type plasmonic chip.

A bright spot is observable in the center of Bull's eye plasmonic pattern with a fluorescence microscope due to the plasmonic nanoantenna effect. In this effect, a propagating wave of surface plasmon resonance concentrates in the center. This study focused on the relationship between the center structure of Bull's eye pattern and the nanoantenna effect in four fabricated Bull's eye-type plasmonic chips with centers of different sizes (full- or half-pitch diameter) and shapes (convex or concave). The fluorescence intensity of the fluorescent nanoparticles adsorbed to these plasmonic chips was measured with an upright-inverted microscope to evaluate the plasmonic chip enhancement factor composed of the product of the excitation and emission enhancement and individual factors. When the emission enhancement factor was investigated under nonresonance excitation conditions, by the disappearance of a bright spot, excitation enhancement was found to contribute to the plasmonic nanoantenna effect. The concave Bull's eye structure with a half-pitch diameter demonstrates the highest nanoantenna effect due to the formation of a larger constructive wave in the superposition of the diffraction wave of incident light under resonance conditions. In addition, the electromagnetic field intensity simulated by discrete dipole approximation agrees with the microscopy results. Overall, the results indicate that the plasmonic nanoantenna effect could be controlled depending on the resonance condition and center structure.

Real-time fluorescence measurement of spontaneous activity in a high-density hippocampal network cultivated on a plasmonic dish.

High-density cultured neuronal networks have been used to evaluate synchronized features of neuronal populations. Voltage-sensitive dye (VSD) imaging of a dissociated cultured neuronal network is a critical method for studying synchronized neuronal activity in single cells. However, the signals of VSD are generally too faint-that is, the signal-to-noise ratio (S/N) is too low-to detect neuronal activity. In our previous research, a silver (Ag) plasmonic chip enhanced the fluorescence intensity of VSD to detect spontaneous neural spikes on VSD imaging. However, no high-density network was cultivated on the Ag plasmonic chip, perhaps because of the chemical instability of the Ag surface. In this study, to overcome the instability of the chip, we used a chemically stable gold (Au) plasmonic dish, which was a plastic dish with a plasmonic chip pasted to the bottom, to observe neuronal activity in a high-density neuronal network. We expected that the S/N in real-time VSD imaging of the Au plasmonic chip would be improved compared to that of a conventional glass-bottomed dish, and we also expected to detect frequent neural spikes. The increase in the number of spikes when inhibitory neurotransmitter receptors were inhibited suggests that the spikes corresponded to neural activity. Therefore, real-time VSD imaging of an Au plasmonic dish was effective for measuring spontaneous network activity in a high-density neuronal network at the spatial resolution of a single cell.

Microscopic Study on Excitation and Emission Enhancement by the Plasmon Mode on a Plasmonic Chip.

Excitation and emission enhancement by using the plasmon mode formed on a plasmonic chip was studied with a microscope and micro-spectroscope. Surface plasmon resonance wavelengths were observed on one-dimensional (1D) and two-dimensional (2D) plasmonic chips by measuring reflection and transmission spectra, and they were assigned to the plasmon modes predicted by the theoretical resonance wavelengths. The excitation and emission enhancements were evaluated using the fluorescence intensity of yellow-green fluorescence particles. The 2D grating had plasmon modes of kgx45(2) (diagonal direction with m = 2) in addition to the fundamental mode of kgx(1) (direction of a square one side) in the visible range. In epifluorescence detection, the excitation enhancement factors of kgx(2) on the 1D and 2D chips were found to be 1.3-1.4, and the emission enhancement factor of kgx45(2) on the 2D chip was 1.5-1.8, although the emission enhancement was not found on the 1D chip. Moreover, enhancement factors for the other fluorophores were also studied. The emission enhancement factor of kgx(1) was shown to depend on the fluorescence quantum yield. The emission enhancement of 2D was 1.3-fold larger than that of 1D considering all azimuth components, and the 2D pattern was shown to be advantageous for bright fluorescence microscopic observation.

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.

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.

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 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.

Spontaneous emission control of CdSe/ZnS nanoparticle monolayer in polymer nanosheet waveguide assembled on a one-dimensional silver grating surface.

We present spontaneous emission control of a core-shell CdSe/ZnS nanoparticle array assembled with polymer ultrathin films consisting of polymer nanosheets on a silver grating substrate, which served as a unique and simple photonic cavity. The grating-coupled waveguide modes enabled 10(3) order luminescence enhancement and one-fourth spectral narrowing. The light emission from a CdSe/ZnS nanoparticle array can be controlled by tuning the film thickness of hybrid polymer nanoassemblies, which provides multiple emission performance with good tuning ability from red to green at low-power continuous wave laser excitation (∼µW).

Zinc oxide-coated plasmonic chip modified with a bispecific antibody for sensitive detection of a fluorescent labeled-antigen.

A plasmonic biosensor chip of silver-coated PMMA grating with a zinc oxide (ZnO) overlayer is fabricated for surface plasmon field-enhanced fluorescence (SPF) detection of Cy5-labeled green fluorescent protein (GFP). A bispecific antibody (anti-GFP x anti-ZnO antibody) prepared in our lab is densely immobilized on the sensor chip for GFP detection. The sensitivity of the plasmonic biosensors is improved due to densely packed antibodies and ZnO-coating that suppresses nonspecific protein adsorption and fluorescent quenching. With the ZnO-coated plasmonic chip, Cy5-labeled GFP of 10 pM can be detected through SPF. This sensitivity is 100 higher compared with the normal fluorescent detection on a ZnO-coated glass slide.

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.

Fluorescence and metal-ion recognition properties of acetylacetone-based ligands.

A new acetylacetone-based ligand (Ligand 1) bearing pyrene unit was synthesized and the fluorescence and metal-ion recognition properties were investigated in the presence of a different metal ion in acetonitrile solution. The fluorescence emission of Ligand 1 was strongly quenched by complexation with Cu(2+) while no significant change was observed in the presence of other metal ions. The metal-ion selectivity changed from Cu(2+) to Ni(2+) when glycine moiety (-NH-CH2-CO-) was introduced into Ligand 1 as spacer. This result implies that hydrogen-bonding induced by the glycine moiety affects the binding ability of ligands to metal ions, resulting in change of the metal-ion selectivity.

Plasmonic coloration of silver nanodome arrays for a smartphone-based plasmonic biosensor.

In this study, the utility of plasmonic coloration on silver nanodome arrays for sensitive and quantitative detection of biomolecules using a smartphone-based sensor is proposed. In particular, a quantitative analysis of DNA hybridization was achieved using the hue angle in the HSV color space obtained from a photograph of a sensing spot taken using a smartphone camera. Silver and gold nanodome arrays consisting of a polystyrene bead layer covered with a thin metal film can be created over a large area by a bottom-up fabrication process. The metal nanodome arrays exhibited unique colorations which can be tuned by the dome diameter ϕ, metal species, and refractive index of the surrounding medium. The measurement of the bulk refractive index sensitivity revealed that the Ag nanodome with ϕ = 500 nm can provide the highest sensitivity of up to 588 nm per refractive index unit. The detection of DNA hybridization was performed by using a bimetallic nanodome consisting of silver and thin gold overlayers and DNA modified gold nanoparticles (AuNPs) for enhancing the sensor signals. Upon the immobilization of AuNPs, the Ag nanodome (ϕ = 200 nm) exhibited a large shift in the resonance wavelength accompanied by a dramatic change in coloration. The analysis of detection sensitivity of DNA hybridization using a model system revealed that colorimetric detection based on hue can be used for the quantitative detection of biomolecules in the same manner as the spectroscopic method with a few pM level of detectable concentration.

Optical microscopic observation of fluorescence enhanced by grating-coupled surface plasmon resonance.

On the substrate carrying a sub-wavelength grating covered with a thin metal layer, a fluorescent dye-labeled cell was observed by fluorescence microscope. The fluorescence intensity was more than 20 times greater than that on an optically flat glass substrate. Such a great fluorescence enhancement from labeled cells bound to the grating substrate was due to the excitation by grating coupled surface plasmon resonance. The application of a grating substrate to two-dimensional detection and fluorescence microscopy appears to offer a promising method of taking highly sensitive fluorescence images.

Optimal Structure of a Plasmonic Chip for Sensitive Bio-Detection with the Grating-Coupled Surface Plasmon-Field Enhanced Fluorescence (GC-SPF).

Surface plasmon field-enhanced fluorescence (SPF) has been one of the powerful tools for biosensors and bioimaging. A wavelength-scale periodic structure coated with a thin metal film is called a plasmonic chip, and it can provide SPF. SPF of Cy5-streptavidin (Cy5-SA) was measured on a biotinylated plasmonic chip with a grating of 480 nm-pitch. The optimal structure of a plasmonic sensor-chip was designed for improving detection sensitivity. The silver film thickness dependence of the SPF intensity was measured under the irradiation of the top panel of a sensor chip. Furthermore, the dependence of the SPF intensity on the distance from the metal surface was also investigated. The optimal structure for the largest fluorescence enhancement factor was 150 nm-thick silver and 10 nm-thick SiO2 layers due to the enhanced electric field (excitation field), the surface plasmon coupled emission (SPCE), and the interference effect with reflected light. The largest enhancement factor was found to be 170-fold. Furthermore, not only the largest fluorescence intensity but also stable lower background noise were found to be essential for higher-sensitive detection.

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.

Oriented attachment-based assembly of dendritic silver nanostructures at room temperature.

How particles aggregate into an interesting dendritic structure has been the object of research for many years because of its importance in understanding physical processes involved and in designing novel materials. In this work, we for the first time describe an oriented attachment-based assembly mechanism for formation of different types of dendritic silver nanostructures at room temperature. It is found that the concentration of both AgNO(3) and p-aminoazobenzene (PA) molecules has a significant effect on the formation and growth of these novel nanostructures. Characterization by transmission electron microscopy (TEM) clearly shows that the dendritic silver nanostructures can be obtained through the preferential oriented growth along a crystallographically special direction. Interestingly, we observe that the oriented attachment at room temperature can also take place between relatively large single-crystalline silver particles with a diameter range from 20 to 60 nm, which may provide a new possibility for the design of novel metal nanostructures by using large metal nanoparticles as building blocks at room temperature. Moreover, a surface-enhanced Raman scattering (SERS) technique is used to investigate the role of PA molecules during the growth of the dendritic silver nanostructures.

Mismatching base-pair dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy.

Two single-stranded DNAs consisting of complementary base pairs except for one mismatching base pair (MM1) can form double-stranded DNA by molecular recognition. This type of duplex is not as stable as that formed by MM0. In order to add to a better understanding of the physical mechanism of the hybridization and dissociation processes at sensor (chip) surfaces, we studied the kinetics of the MM1 hybridization by surface plasmon fluorescence spectroscopy. Target DNA strands labelled with a fluorescent molecule Cy5 at the 5' end and hybridizing with the surface-attached probe DNA can be excited by the strong optical field of a surface plasmon resonance mode. The emitted fluorescence can be detected with high sensitivity. The affinity of a duplex was found to depend on the chemical nature, i.e. G-G, G-T etc., and on the position of the mismatching base pair along the 15mer duplex.

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.

A plasmonic chip-based bio/chemical hybrid sensing system for the highly sensitive detection of C-reactive protein.

A synthetic polymer ligand-grafted plasmonic chip was fabricated and demonstrated a highly sensitive detection of C-reactive protein (CRP) by grating-coupled surface plasmon field-enhanced fluorescence. Poly(2-methacryloyloxyethyl-phosphorylcholine) was used as a CRP-specific polymer ligand layer and was grafted onto the plasmonic chip using surface-initiated controlled/living radical polymerization (limit of detection: ca. 10 pM).