Surporting information
Duty ratio-dependent fluorescence enhancement through
surface plasmon resonance in Ag-coated gratings
Xiaoqiang Cui a, Keiko Tawa*a, Hironobu Hori a, and Junji Nishiib
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and
Technology (AIST), Ikeda, Osaka 563-8577, Japan
b
Photonics Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), Ikeda, Osaka 563-8577, Japan
* E-mail address: tawa-keiko@aist.go.jp
a
Experimental
Fabrication of grating SiO2 substrate
Sub-wavelength grating structures on SiO2 substrates were constructed by previously
reported two-laser-beam interference method followed by dry etching
[1]
. All
substrates were cleaned by sonicating in a 1% Hellmanex (Hellma, Mullheim,
Germany) solution for 20 min and thoroughly rinsed by fresh Milli-Q water and
ethanol, finally dried completely under air flow. Cleaned SiO2 plates were first
spin-coated with about 1 m thickness of positive photoresist (Tokyo Ohka Kogyo
Co., Ltd., TDMR-AR80). The photoresist pattern was then formed by exposing to an
interference beam of a He-Cd laser (Kimmon Electric, 1K3501R-G) with 325 nm
wavelength and a power density of about 70 W/cm2 for 100 s. Duty ratio of the
grating was controlled by irradiation time. The SiO2 surface was then etched by dry
etching (ULVAC, NLD-500). Grating depth was controlled by changing etching time
according to the relation of about 2 nm per second. The residual photoresist was then
removed by acetone washing and then oxygen plasma treatment. (Yamato,
RFG-500A).
Coating with metal layers
The SiO2 gratings were consequently coated with thin layers of Cr(1 nm)/Ag(200
1
nm)/Cr(1 nm)/SiO2(20 nm) by RF sputtering deposition (Rikensya, specially made).
In order to improve the adhesion of Ag to SiO2, a Cr layer of less than 1-nm was
deposited. After which, 200-nm silver layer was deposited followed by another layer
of Cr, and then 20nm layer of SiO2. Therefore, the covered layer consists of
Cr/Ag/Cr/SiO2 with the total thickness of about 222 nm. After deposition, the surface
was immediately treated with 3-aminopropyltriethoxysilane (APTES, 1% vol)
aqueous solution at 40C for 2 hours and then thoroughly rinsed by Milli-Q water and
ethanol, with which the surface was modified with –NH2 groups for the further
functionalization of biotin-poly(ethylene glycol)-Carbonate-NHS ester (NOF,
SUNBRIGHT BI-050TS).
Scheme SI. A schematic diagram of the angular scanning measurement setup for reflectivity (SPR) and
fluorescence intensity (SPFS).
SPR-SPFS measurement
SPR-SPFS measurement was performed using originally constructed set up described
previously with slightly modification (Scheme SI)[2]. A He-Ne laser beam (632.8 nm,
1 mm in diameter) serves as incident light on substrates for SPR-SPFS excitation. An
optical chopper (used also as the reference for the lock-in amplifier) and two
polarizers were used for intensity and polarization control respectively. During
measurement, the reflected light was monitored by photo diode through a -2
goniometer
controlling.
The
fluorescence
emission
was
monitored
by a
photomultiplier (after passing through an appropriate lens, 1% and 10%-ND filter, and
a narrow band interference filter, =6705 nm) mounted on the goniometer unit at a
fixed angle of 55 degree. Therefore, the fluorescence enhanced by reverse coupling
mode was not included in the detection.
2
Finite difference time domain (FDTD) simulation
FDTD was used to simulate the electric field distribution around the grating surfaces.
The cross-section profile for the simulation unit was drawn according to the SPM data.
To explore the effect of different duty ratios, the pitch (400 nm), depth (20 nm), and
trapezoidal slope angle (25 degrees) were all kept constant. The calculations were
carried out for incident light with a wavelength of 632.8 nm. The periodic boundary
was perpendicular to the grating surface, while a perfectly matched absorbing
boundary was parallel to the grating surface.
Fluorescence microscopy
Fluorescence microscopy observation was taken on an upright microscope (BX51WI,
Olympus, Tokyo, Japan) equipped with halogen (12 V 100WHAL-L, Olympus) and
Hg lamp as well as an x10 objective lens (NA 0.30). The fluorescence images were
observed by selecting a Cy5 filter with a CCD camera (Andor Technology, iXonEM+).
During the series of measurements, illumination intensity (from Hg lamp), exposure
time of 0.03 second, and CCD camera gain were all kept constant. We first patterned
the grating surfaces with tape to make the comparison of fluorescence intensities
easier. After coating, the tape was removed and the surfaces were modified with
Cy5-labeled streptavidin, and then the samples were observed using a commercially
available microscope.
Fluorescence enhancement on gratings with different depth:
Fig. S1. SPR curves in air measured using grating with depth of 10 nm, 20 nm and 30 nm, respectively. Duty ratio
for each depth is the optimized condition.
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Table S1.
Enhancement factors from gratings with different depth under
microscopic observation
Grating depth
10 nm
20 nm
30 nm
Enhancement factor
10
30
28
Highest enhancement is obtained in 20nm depth grating. All experimental conditions
are same as that in Fig. 4.
Dispersion relationship and resonance angle
The angle-dependent effect of the enhanced fluorescence was explained by the
dispersion relation.
The wavenumber vectors of surface plasmon polaritons, incident
light (of a propagated component), and a grating denoted as kspp, k0x, and kg, can be
used to determine the dispersion relationship for Ag-coated gratings as:
kspp = k0x + m kg.
(m = 1 ,2, - - - )
(1)
Here, Eq. (1) can be expressed by:
/c ((Ag ∙PBS)/( Ag + PBS))1/2 = /c sin  m2/
(2)
in which , c, , , and  are the angular frequency, speed of light, the complex
dielectric constant for a material expressed by the subscript, the incident angle, and
the pitch of the grating, respectively.
Detailed discussion of the duty-ratio dependence of the fluorescence
enhancement
In general, the electric field intensity can be expressed as a function of both
Fresnel’s transmission coefficient described by the dielectric constant and the
reflectivity (R), i.e., 1–R, at a given resonance angle on the GC-SPR[3]. Therefore, the
duty ratio dependence of the enhancement of the fluorescence can be interpreted by
theoretically analyzing the relationship between the coupling efficiency, i.e., R, and
the grating surface profile, including the duty ratio.
Giannattasio et al.[4] reported the duty ratio dependence of the transmitted
diffracted intensity (T–1) using a grating-assisted prism-coupled SPR. Taking into
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account the fundamental difference between our work and this paper, including the
optical geometry for the light–SPP coupling, the physical parameters to be detected,
and the coupling modes (whether reverse coupling process was included or not), a
duty ratio dependence of T–1 through the prism via SPP coupling can help us to
theoretically interpret our results. In the work of Giannattasio et al., the profile of a
periodic corrugation was expressed in terms of a Fourier expansion. The value of T–1
increased with the square of the grating amplitude and provided the largest value
using a first-order approximation for a ratio of 0.50. SPR coupling is also known to be
correlated with the effect of the duty ratio on the grating profile amplitude. For the
largest amplitude, a duty ratio of 0.50 is considered to provide the smallest value of R,
the strongest electric field, and consequently, the most enhanced fluorescence
intensity in our study.
Reference
[1] K. Kintaka, J. Nishii, A. Mizutani, H. Kikuta, H. Nakano, Opt. Lett. 2001, 26, 1642-1644. J. Nishii, K.
Kintaka, Y. Kawamoto, A. Mizutani, H. Kikuta, J. Ceram. Soc. Jpn. 2003, 111, 24-27.
[2] K. Tawa, H. Hori, K. Kintaka, K. Kiyosue, Y. Tatsu, J. Nishii, Opt. Express 2008, 16, 9781-9790.
[3] H. Raether, Surface plasmons on smooth and rough surfaces and on gratings. (Springer, Berlin,
1988).
[4] A. Giannattasio, I. R. Hooper, W. L. Barnes, Opt. Commun. 2006, 261, 291-295.
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