Duty ratio-dependent fluorescence enhancement through surface plasmonresonance in Ag-coated gratingsXiaoqiang Cui, Keiko Tawa, Hironobu Hori, and Junji Nishii Citation: Applied Physics Letters 95, 133117 (2009); doi: 10.1063/1.3238562 View online: http://dx.doi.org/10.1063/1.3238562 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/95/13?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Sensing properties of infrared nanostructured plasmonic crystals fabricated by electron beam lithography andargon ion milling J. Vac. Sci. Technol. B 30, 06FE02 (2012); 10.1116/1.4767274 Surface profile dependence of the photon coupling efficiency and enhanced fluorescence in the grating-coupled surface plasmon resonance J. Appl. Phys. 107, 114702 (2010); 10.1063/1.3408446 Enhancement of diffraction for biosensing applications via Bloch surface waves Appl. Phys. Lett. 91, 253125 (2007); 10.1063/1.2826545 Periodic nanohole arrays with shape-enhanced plasmon resonance as real-time biosensors Appl. Phys. Lett. 90, 243110 (2007); 10.1063/1.2747668 Enhancement of ultraviolet lasing from Ag-coated highly disordered ZnO films by surface-plasmon resonance Appl. Phys. Lett. 90, 231106 (2007); 10.1063/1.2746940

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Duty ratio-dependent fluorescence enhancement through surface plasmonresonance in Ag-coated gratings

Xiaoqiang Cui,1 Keiko Tawa,1,a� Hironobu Hori,1 and Junji Nishii21Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology(AIST), Ikeda, Osaka 563-8577, Japan2Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),Ikeda, Osaka 563-8577, Japan

�Received 1 May 2009; accepted 3 September 2009; published online 1 October 2009�

One-dimensional gratings with different duty ratios were designed and implemented for enhancedfluorescence detection and imaging. Verified by finite difference time domain simulations, ourresults showed that the enhancement strongly depended on the duty ratio of the land width to pitchof the grating structure. The maximum enhancement factor was achieved when the duty ratio wasequal to 0.50 in our trapezoidal gratings with pitch=400 nm and depth=20 nm. Such a facilegrating mold will exert a considerable influence on microarray biosensors and fluorescencemicroscopy. © 2009 American Institute of Physics. �doi:10.1063/1.3238562�

Fluorescence detection is rapidly becoming a leadingmethodology used in life sciences, and has been widely usedin biological and medical research. Efforts in improving thefluorescence output to achieve lower detection limits orbrighter fluorescence images with a high contrast have led toa powerful technique called surface-enhanced fluorescence�SEF�.1–5 Until now, most work on SEF has focused on lo-calized surface plasmon resonance �SPR�-based nanopar-ticles or nanoislands, which still exhibit quenching and poorreproducibility.6–9 On the other hand, fluorescence enhance-ment using propagated SPR has been developed for reliablebiospecific interaction analysis.8,10,11 In overcoming thedrawback of requiring complicated optical setups inKretschmann geometry, grating-coupled SPR �GC-SPR�, inwhich the resonance angle can be controlled below 10° bythe pitch of a fabricated grating,12–14 is an ideal candidate toenhance fluorescence for fluorescence microscopy and mi-croarray readers. Surprisingly, only a few works have beenpublished exploiting GC-SPR for enhanced fluorescence ob-servations. Recently, GC-SPR from Alq3 or a dielectric grat-ing structure composed of a metal layer was utilized for or-ganic light-emitting diode devices and biosensors.15,16 Ourgroup has developed a technique for the detection of en-hanced fluorescence images of proteins and cells on silver-coated SiO2 gratings using a fluorescence microscope.17 Al-though we have shown that the detail of the grating profileinfluences the enhancing efficiency,18 this dependency is notfully understood. In this letter, the fluorescence enhancementefficiency was investigated experimentally and theoreticallyas a function of the duty ratio.

The gratings had a pitch=400 nm and a depth=20 nm,and were fabricated on SiO2 substrates using a two-beamlaser interference method associated with the dry etchingprocess developed in our previous report.19,20 The geometryof the gratings could be varied over a wide range by chang-ing the processing parameters. By increasing the irradiationtime at low laser power density, the duty ratio graduallyshrank from 0.72 to 0.33. The duty ratio is defined as theland width divided by the grating pitch. Three typical grating

surfaces characterized by an atomic force microscopy �AFM�are shown in Fig. 1. Thin layers of Cr /Ag /Cr /SiO2 weresputtered on the substrates sequentially. The pitch and thegroove depth were unchanged after the coating process, butthe duty ratio �e.g., 0.43–0.50� and the surface roughnessincreased �Figs. 1�a� and 1�b�� due to accumulation. It isworth noting that the land width for the duty ratio calculationwas obtained at the half-depth of the trapezoidal grating �thered bar in Fig. 1�c��. The coated substrates were immediatelymodified by 3-aminopropyltriethoxysilane for further func-tionalization of biotin.

The incident angle-dependent reflectivity and fluores-cence intensity were measured to characterize the couplingefficiency and the enhancement of the excitation field, whichmeans that a lower reflectivity was achieved for a given reso-nance angle, with a consequent stronger enhancement of thefluorescence. The fluorescent dye-labeled protein, Cy5-streptavidin, was used as the probe to evaluate the GC-SPR-enhanced fluorescence spectra �GC-SPFS�. Details of the ex-

a�Electronic mail: tawa-keiko@aist.go.jp.

FIG. 1. �Color online� AFM images of SiO2 gratings fabricated withdifferent duty ratios, �a� before and �b� after coating withCr�1 nm� /Ag�200 nm� /Cr�1 nm� /SiO2�20 nm�. After coating, the dutyratio increased from 0.43 to 0.50. �c� AFM cross-section curves obtainedfrom �a� clearly show the difference in duty ratio. The red bar in �c� repre-sents the land width used to calculate the duty ratio.

APPLIED PHYSICS LETTERS 95, 133117 �2009�

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perimental methods and setup are described in supportinginformation �Scheme S1�.21 The reflectivity and fluorescenceresponse versus the incident angle were measured for thegratings and the control substrate surfaces, respectively �Fig.2�a��. On the grating surface, the intensity of the fluorescence�curve F-1� from the Cy5-streptavidin showed a maximum atthe resonance angle of 8.5° from the dip in reflectivity �curveR-s�. However, the fluorescence intensity was constant�curves F-2 and F-3� on both coated and bare glass slides, asno SPR reflectivity dip was observed �data not shown�. Thisindicates that the enhancement of the fluorescence on thegrating structure was due to the enhanced electric field fromthe GC-SPR. Using bare glass as a control surface, the en-hancement factor �EF� was calculated and plotted togetherwith the reflectivity versus duty ratios �shown in Fig. 2�b��. Itcan be seen that the optimal duty ratio of 0.43 was obtainedfor a maximum EF=40, which is much higher than previ-ously obtained �24 and 30 times17�. The AFM results show inFig. 1 indicated that the duty ratio of 0.43 had increased to0.50 with a trapezoidal shape after coating. This optimal con-dition was in agreement with the theoretical calculationsfrom a dielectric grating waveguide coupler,22 and the ex-perimental results of light transmitted in the first diffractedorder in the optical geometry of a grating-assisted prism-coupled SPR.23

The finite difference time domain �FDTD� was used tosimulate the electric field around grating surfaces with dif-ferent duty ratios. The cross-section profile for the simulationunit is shown in Fig. 3�a�. As expected, the electric-fieldintensity ��E�2� varied according to the incident angle andreached maximum around 9° �Fig. 3�b��, which is in agree-ment with the experimental results of 8.5° in Fig. 2 and withthe calculation results of 10.9° obtained from the dispersion

relationship.21 At this resonance angle, a map pattern is moreuseful to illustrate the detailed distribution of the electricfield intensity �Fig. 3�c��. The field intensity was concen-trated on the vertex angle area of the trapezoidal shapedsurface. A similar phenomenon has also been observed inmany grating studies and in nanocavity-based plasmonicstructures.14,24,25 On comparing the traces in Fig. 3�c�, wecan conclude that a grating duty ratio of 0.50 after coatingwill result in the strongest fluorescence enhancement. Thisduty ratio dependant behavior could also be interpreted byexpressing a periodic corrugation in terms of a Fourier ex-pansion using a first-order approximation such as the analy-sis of a duty ratio dependence of transmitted diffractedintensity.23

The fluorescence EF from gratings with different dutyratios was quantified using a fluorescence microscope. Atypical bright field and fluorescence image from the sub-strates is shown in Figs. 4�a� and 4�b�. Using our patterningstrategy, the surfaces were divided into three parts, coatedgrating, coated flat glass, and bare glass, indicated as areas I,II, and III, respectively. The Cy5 fluorescence was only ob-served on the grating surface �area I�, indicating that en-hancement only occurred on this surface compared with theother two surfaces �areas II and III�. Using the average fluo-rescence intensity on bare glass �I0� as a control surface, theenhanced factor was calculated using the ratio I / I0 and thedata were plotted versus the duty ratio, as shown in Fig. 4�c�.It can be seen that the EF under the microscope was alsoduty ratio-dependent with a variation between 19 and 30times. The maximum enhancement of 30 times at a duty ratioof 0.43 was lower than that observed using angular scanningfluorescence detection �40 times� as discussed above. Thismay be reasonable if we consider the difference between thetwo measuring techniques. Under microscope observation,the incident angle within the field of view covers a range of0°–17°, which means that light illuminates not only from theresonance angle �8.5°� but also from other angles that areunable to excite to the highest level of enhancement. On theother hand, in the angular scanning measurements, the EFwas always calculated at the resonance angle of highest en-

FIG. 2. �Color online� �a� Reflectivity �SPR� and fluorescence intensity�SPFS� vs incident angle: SPR �R-s� and SPFS on Cy5/Ag/grating �F-1�,Cy5/Ag/plate glass �F-2�, Cy5/glass �F-3�, and bare glass �F-4�. �b� The dipin reflectivity of the SPR �blue� and the fluorescence enhancement �red� vsduty ratio for a 20-nm depth grating. The EF was calculated from EF= �IF1− IF4� / �IF3− IF4�, where IFi presents the fluorescence intensity at theresonance angle indicated by the dashed line in �a�.

FIG. 3. �Color online� �a� Grating profile for a drawing unit before, and aftercoating for the FDTD simulations. �b� Calculated electric-field intensity �E2�vs incident angle plots for duty ratios of 0.40, 0.45, 0.50, 0.55, and 0.60 aftercoating. �c� Map patterns for the calculated electric field intensities on thegratings �0.40, 0.50, and 0.60 after coating� using p-polarized light incidentat their resonance angle.

133117-2 Cui et al. Appl. Phys. Lett. 95, 133117 �2009�

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hancement. Furthermore, p-polarized light was used for theangular scanning measurements, while the nonpolarized lightused in the microscope could not always effectively couplewith the surface plasmon polaritons. The differences in cou-pling modes may also lead to complicated discussion in EFs.For instance, whether an enhanced emission based on thereverse coupling should be considered or not, as the reso-nance angle of reverse coupling is expected from theoreticaldispersion relation to be 13° which are included within 0°–17° used in microscopy. The enhancement effect was alsoinvestigated for different depth gratings. The results showedthat under the optimal duty ratio, a grating with a depth of 20nm has the best SPR coupling, and consequently the highestfluorescence enhancement �Fig. S1 and Table S1�.21

In conclusion, surface relief gratings with different dutyratios were fabricated and applied in enhanced fluorescencedetection and microscopic observations. Experimental dataand FDTD calculations showed that the maximum enhancedfluorescence was obtained under optimized conditions with aduty ratio of 0.43 �0.50 after coating�. Our investigation us-

ing Cy5-protein patterns under a general microscope prom-ises great potential for these substrates in both biosensingand biological imaging.

This work was supported by KAKENHI �Grant-in-Aidfor Scientific Research� No. 19049016 in the priority area“Strong Photons-Molecules Coupling Fields �No. 470�” fromthe Ministry of Education, Culture, Sports, Science andTechnology of Japan.

1E. Fort and S. Gresillon, J. Phys. D 41, 013001 �2008�.2K. S. Phillips and Q. Cheng, Anal. Bioanal. Chem. 387, 1831 �2007�.3Y. Fu, J. Zhang, and J. R. Lakowicz, J Fluoresc. 17, 811 �2007�.4R. M. Bakker, H. K. Yuan, Z. T. Liu, V. P. Drachev, A. V. Kildishev, V. M.Shalaev, R. H. Pedersen, S. Gresillon, and A. Boltasseva, Appl. Phys. Lett.92, 043101 �2008�.

5H. Choumane, N. Ha, C. Nelep, A. Chardon, G. O. Reymond, C. Goutel,G. Cerovic, F. Vallet, C. Weisbuch, and H. Benisty, Appl. Phys. Lett. 87,031102 �2005�.

6T. D. Corrigan, S. H. Guo, H. Szmacinski, and R. J. Phaneuf, Appl. Phys.Lett. 88, 101112 �2006�.

7Z. Y. Wang, Z. J. Chen, Z. H. Lan, X. F. Zhai, W. M. Du, and Q. H. Gong,Appl. Phys. Lett. 90, 151119 �2007�.

8F. Xie, M. S. Baker, and E. M. Goldys, Chem. Mater. 20, 1788 �2008�.9J. Zhang, Y. Fu, M. H. Chowdhury, and J. R. Lakowicz, Nano Lett. 7,2101 �2007�.

10K. Tawa, D. F. Yao, and W. Knoll, Biosens. Bioelectron. 21, 322 �2005�.11K. Tawa and W. Knoll, Nucleic Acids Res. 32, 2372 �2004�.12H. Raether, Surface Plasmons on Smooth and Rough Surfaces and on

Gratings �Springer, New York, 1988�.13W. Knoll, Annu. Rev. Phys. Chem. 49, 569 �1998�.14E. Popov, N. Bonod, and S. Enoch, Opt. Express 15, 4224 �2007�.15Y. J. Hung, I. I. Smolyaninov, C. C. Davis, and H. C. Wu, Opt. Express

14, 10825 �2006�.16N. F. Chiu, C. Yu, S. Y. Nien, J. H. Lee, C. H. Kuan, K. C. Wu, C. K. Lee,

and C. W. Lin, Opt. Express 15, 11608 �2007�.17K. Tawa, H. Hori, K. Kintaka, K. Kiyosue, Y. Tatsu, and J. Nishii, Opt.

Express 16, 9781 �2008�.18H. Hori, K. Tawa, K. Kintaka, J. Nishii, and Y. Tatsu, Opt. Rev. 16, 216

�2009�.19K. Kintaka, J. Nishii, A. Mizutani, H. Kikuta, and H. Nakano, Opt. Lett.

26, 1642 �2001�.20J. Nishii, K. Kintaka, Y. Kawamoto, A. Mizutani, and H. Kikuta, J. Ceram.

Soc. Jpn. 111, 24 �2003�.21See EPAPS supplementary material at http://dx.doi.org/10.1063/

1.3238562 for experimental methods, dispersion relation for SPR anglecalculation and depth effect on enhancement.

22A. Tamir and S. T. Peng, Appl. Phys. 14, 235 �1977�.23A. Giannattasio, I. R. Hooper, and W. L. Barnes, Opt. Commun. 261, 291

�2006�.24J. Li, H. Iu, W. C. Luk, J. T. K. Wan, and H. C. Ong, Appl. Phys. Lett. 92,

213106 �2008�.25J. Li, H. Iu, J. T. K. Wan, and H. C. Ong, Appl. Phys. Lett. 94, 033101

�2009�.

FIG. 4. �Color online� Microscopic observation for �a� bright field and �b�fluorescence images of patterned grating structure with the following areas:�i� metal-coated grating, �II� metal-coated glass, and �III� bare glass. Theentire surface was modified using Cy5-streptavidin. �c� Fluorescence inten-sity �normalized to the bare glass I0� for different duty ratios, where areas IIand III represent the conditions for coated and bare glass, respectively. Thescale bar represents 100 �m.

133117-3 Cui et al. Appl. Phys. Lett. 95, 133117 �2009�

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