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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 12 — Jun. 4, 2012
  • pp: 12850–12859
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Enhanced fluorescence in a nanoporous waveguide and its quantitative analysis

Yong Fan, Kazuhiro Hotta, Akira Yamaguchi, and Norio Teramae  »View Author Affiliations


Optics Express, Vol. 20, Issue 12, pp. 12850-12859 (2012)
http://dx.doi.org/10.1364/OE.20.012850


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Abstract

Fluorescence behavior was examined for fluorophore-labeled protein (BSA-AF) adsorbed on the nanopore surface of a nanoporous waveguiding film. The waveguiding film has a bilayer structure of a porous anodic alumina (PAA) layer on a metallic aluminum (Al) layer, and this structure allows efficient interaction of fluorophores entrapped in the nanoporous waveguiding film with a hotspot of the enhanced electromagnetic field of the waveguide modes. Fluorescence response of BSA-AF depends on the enhanced field within the waveguiding film and the enlarged adsorbed amount in the PAA layer where most of the light is confined. Enhancement of the field in the waveguiding film can be controlled by the refractive index of the PAA layer and enlargement of the pore size efficiently affects the enhancement of the fluorescence response. Compared to the film without a PAA layer, the PAA/Al film exhibits more than 140-fold larger fluorescence response due to the large adsorption capacity of the PAA nanopores and the enhanced field formed by the waveguide modes in the PAA layer with a low refractive index.

© 2012 OSA

1. Introduction

Fluorescence has been widely used in optical devices, biomolecule labeling, label-free bioassays, and cellular imaging. Although fluorescence detection is recognized as a sensitive analytical technique, continuing efforts have been made to increase its sensitivity by developing new efficient fluorophores or using the interaction of fluorophores with nanostructured metallic surfaces which is known as metal-enhanced fluorescence or surface-enhanced fluorescence [1

1. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008). [CrossRef]

, 2

2. J. R. Lakowicz, K. Ray, M. Chowdhury, H. Szmacinski, Y. Fu, J. Zhang, and K. Nowaczyk, “Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy,” Analyst (Lond.) 133(10), 1308–1346 (2008). [CrossRef] [PubMed]

]. Such fluorescence enhancement from excited fluorophores is obtained by modifications of the photonic mode density (PMD), i.e., the density of possible electromagnetic decay channels [3

3. W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt. 45(4), 661–699 (1998). [CrossRef]

, 4

4. F. D. Stefani, K. Vasilev, N. Bocchio, F. Gaul, A. Pomozzi, and M. Kreiter, “Photonic mode density effects on single-molecule fluorescence blinking,” New J. Phys. 9(2), 21 (2007). [CrossRef]

]. Modifications of PMD by placing fluorophores in proximity to metallic nanostructures lead to the increased radiative decay rate, and the increased excitation rate can be achieved by altering the electromagnetic field. Various metallic nanostructures have been used to obtain enhanced fluorescence such as nanoparticles [5

5. J. Zhang and J. R. Lakowicz, “Metal-enhanced fluorescence of an organic fluorophore using gold particles,” Opt. Express 15(5), 2598–2606 (2007). [CrossRef] [PubMed]

, 6

6. Y. Chen, K. Munechika, and D. S. Ginger, “Dependence of fluorescence intensity on the spectral overlap between fluorophores and plasmon resonant single silver nanoparticles,” Nano Lett. 7(3), 690–696 (2007). [CrossRef] [PubMed]

], nanorods [7

7. R. Bardhan, N. K. Grady, J. R. Cole, A. Joshi, and N. J. Halas, “Fluorescence enhancement by Au nanostructures: nanoshells and nanorods,” ACS Nano 3(3), 744–752 (2009). [CrossRef] [PubMed]

, 8

8. Y. Fu, J. Zhang, and J. R. Lakowicz, “Plasmon-enhanced fluorescence from single fluorophores end-linked to gold nanorods,” J. Am. Chem. Soc. 132(16), 5540–5541 (2010). [CrossRef] [PubMed]

], thin metal films [9

9. S. Wedge and W. L. Barnes, “Surface plasmon-polariton mediated light emission through thin metal films,” Opt. Express 12(16), 3673–3685 (2004). [CrossRef] [PubMed]

, 10

10. S. Fang, H. J. Lee, A. W. Wark, H. M. Kim, and R. M. Corn, “Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy,” Anal. Chem. 77(20), 6528–6534 (2005). [CrossRef] [PubMed]

], nanowires [11

11. S.-H. Guo, J. J. Heetderks, H.-C. Kan, and R. J. Phaneuf, “Enhanced fluorescence and near-field intensity for Ag nanowire/nanocolumn arrays: evidence for the role of surface plasmon standing waves,” Opt. Express 16(22), 18417–18425 (2008). [CrossRef] [PubMed]

], nanocavities or nanoholes [12

12. J. P. Hoogenboom, G. Sanchez-Mosteiro, G. Colas des Francs, D. Heinis, G. Legay, A. Dereux, and N. F. van Hulst, “The single molecule probe: Nanoscale vectorial mapping of photonic mode density in a metal nanocavity,” Nano Lett. 9(3), 1189–1195 (2009). [CrossRef] [PubMed]

, 13

13. P.-F. Guo, S. Wu, Q.-J. Ren, J. Lu, Z. Chen, S.-J. Xiao, and Y.-Y. Zhu, “Fluorescence enhancement by surface plasmon polaritons on metallic nanohole arrays,” J. Phys. Chem. Lett. 1(1), 315–318 (2010). [CrossRef]

], and corrugated gratings [14

14. Y.-J. Hung, I. I. Smolyaninov, C. C. Davis, and H. C. Wu, “Fluorescence enhancement by surface gratings,” Opt. Express 14(22), 10825–10830 (2006). [CrossRef] [PubMed]

, 15

15. Y. Jiang, H.-Y. Wang, H. Wang, B.-R. Gao, Y.- Hao, Y. Jin, Q.-D. Chen, and H.-B. Sun, “Surface plasmon enhanced fluorescence of dye molecules on metal grating films,” J. Phys. Chem. C 115(25), 12636–12642 (2011). [CrossRef]

]. On the other hand, it has been reported that fluorescence is strongly quenched if fluorophores are located very close to metal surfaces, though the fluorescence enhancement increases exponentially as the distance between the fluorophores and the metal surfaces decreases [16

16. N. Ganesh, P. C. Mathias, W. Zhang, and B. T. Cunningham, “Distance dependence of fluorescence enhancement from photonic crystal surface,” J. Appl. Phys. 103(8), 083104 (2008). [CrossRef]

]. Accordingly, careful positioning of fluorophores is necessary to get maximum enhancement of their fluorescence near the metal surface. To overcome the need for this accurate positioning, detection of enhanced fluorescence has recently been proposed by using the field of the optical waveguide modes which can be formed by metal-clad waveguiding films [17

17. A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express 14(8), 3512–3527 (2006). [CrossRef] [PubMed]

, 18

18. N. Ganesh, I. D. Block, P. C. Mathias, W. Zhang, E. Chow, V. Malyarchuk, and B. T. Cunningham, “Leaky-mode assisted fluorescence extraction: application to fluorescence enhancement biosensors,” Opt. Express 16(26), 21626–21640 (2008). [CrossRef] [PubMed]

]. Metal-clad waveguides are composed of a thin dielectric layer on a thin semi-transparent metal layer and light is partially confined in the thin dielectric layer [17

17. A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express 14(8), 3512–3527 (2006). [CrossRef] [PubMed]

]. Distribution of light in metal-clad waveguides is within a micron range from the metal surface and it covers a larger area compared to the ca. 100-200 nm range for surface plasmon resonance. Simulation of metal-clad waveguides showed ca. 70-fold increase in the fluorescence intensity compared to the case without metal-clad waveguides [17

17. A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express 14(8), 3512–3527 (2006). [CrossRef] [PubMed]

], and high sensitivity was demonstrated by using a sensor comprised of a dielectric hydrogel layer on a thin Au layer [19

19. C. J. Huang, J. Dostalek, and W. Knoll, “Long range surface plasmon and hydrogel optical waveguide field-enhanced fluorescence biosensor with 3D hydrogel binding matrix: On the role of diffusion mass transfer,” Biosens. Bioelectron. 26(4), 1425–1431 (2010). [CrossRef] [PubMed]

].

In this study, we examined fluorescence enhancement for a planar nanoporous waveguiding (NPWG) film comprised of a porous anodic alumina (PAA) dielectric layer on a metallic aluminum (Al) layer. The electromagnetic field of incident light is mostly confined as an optical waveguide mode within the PAA layer with a thickness of several hundred nanometers, and the field distribution can be simulated by the method reported in the literature [20

20. W. N. Hansen, “Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium,” J. Opt. Soc. Am. 58(3), 380–390 (1968). [CrossRef]

]. In the optimal condition, the field intensity of the waveguide mode was reported to be several hundred-fold higher than that of incident light [17

17. A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express 14(8), 3512–3527 (2006). [CrossRef] [PubMed]

]. The PAA dielectric layer has a high surface area because of its nanoporous structure, which allows more analyte molecules within the sensing dielectric layer compared to the solid planar dielectric films. In addition, the maximum field intensity of the waveguide mode typically appears a hundred nanometers or more away from the metallic surface [20

20. W. N. Hansen, “Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium,” J. Opt. Soc. Am. 58(3), 380–390 (1968). [CrossRef]

], which can efficiently suppress the fluorescence quenching effect by metal. Accordingly, the NPWG composed of a PAA/Al film can serve as a platform for fluorescence detection with an enhanced response. Here, we report on a large fluorescence enhancement due to the intensified field formed in the PAA layer and the large adsorption area of the NPWG film. The relationship between the fluorescence enhancement and the field distribution was examined by measuring angular reflection and angular fluorescence spectra of the NPWG film for adsorption of bovine serum albumin labeled with Alexa fluor® (BSA-AF) onto the pore surface of the PAA waveguiding layer. The enhancement was investigated in detail considering the design parameters of the PAA layer and was quantitatively analyzed by evaluating both the fluorescence intensity and the adsorbed amount of BSA-AF.

2. Materials and methods

2-1. Materials

Square cover glass (25 × 25 × 0.3 mm; Matsunami Glass Ind., Ltd., Osaka, Japan) was used as a substrate for fabrication of the PAA/Al multilayer film. Al wires (99.99%; Nilaco Co., Tokyo, Japan) were used to form Al films on glass substrates by thermal deposition. Milli-Q water (Millipore Corp., Bedford, MA) was used for all experiments. BSA-AF was purchased from Molecular Probes, Inc. (Eugene, OR). Experiments on adsorption of BSA-AF to the waveguiding layer were done in a phosphate buffer solution containing 5 mM KH2PO4 (pH 4.6) to maximize the adsorption of BSA to the PAA surface [21

21. S. Fukuzaki, H. Urano, and K. Nagata, “Adsorption of bovine serum albumin onto metal oxide surfaces,” J. Ferment. Bioeng. 81(2), 163–167 (1996). [CrossRef]

]. A 100 nM BSA-AF solution was prepared by dissolving BSA-AF in the buffer solution. All the other organic solvents and chemical reagents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and were used as received.

2-2. Fabrication of NPWG based on the PAA/Al bilayer film

2-3. Optical setup

Optical characteristics of the NPWG (PAA/Al) film were examined by measuring angular reflection spectra (reflectivity vs. incident angle) and angular fluorescence spectra (fluorescence intensity vs. incident angle) in the Kretschmann configuration as shown in Fig. 2
Fig. 2 Schematic illustration for the fluorescence detection based on the NPWG (PAA/Al) film.
. Radiation from a diode laser (LCS-DTL-364, λ = 472 nm, 3 mW, Laser-Compact Co. Ltd., Moscow, Russia) was modulated by a light chopper (CH-353, NF Corp., Yokohama, Japan) at 640 Hz and s- or p- polarized light was obtained by using a polarizer. The laser beam was directed on the PAA/Al substrate attached to a BK7 equilateral prism via an index matching fluid (immersion oil No. 16242, Cargille-Sacher Lab. Inc., Cedar Grove, NJ) at a certain angle of incidence (θ). Reflected light was detected by a Hamamatsu model S2281 Si PIN photodiode and the signals were processed by a lock-in-amplifier (L74-836, NF Corp., Yokohama, Japan). An analyzer was set in front of the detector to ensure that only one kind of polarized light reached the detector. The reflectivity of the glass substrate was normalized using the intensity of reflected light observed for a bare glass substrate attached to the BK7 prism.

Fluorescence emitted from the NPWG film was collected by using a lens unit composed of a couple of lenses (f = 20 mm, ϕ = 12.7 mm and f = 30 mm, ϕ = 12.7 mm) and a long pass filter (FGL495, Thorlabs, Inc., Newton, NJ). The lens unit was coupled with an optical fiber that guided light to a polychromator (MS3501 with a 300 lines/mm grating, Solar TII, Ltd., Minsk, Belarus) with a CCD detector (iDus DU420A-0E, 1024 × 255 pixels, Andor Technology Plc., Belfast, Northern Ireland). For angular fluorescence spectra, fluorescence intensity was measured against θ at the maximum emission wavelength of BSA-AF (λ = 520 nm). A PDMS flow cell with a cell volume of 2 μL (16 mm × 1.3 mm × 100 μm) was attached to the NPWG substrate and a buffer solution containing 100 nM BSA-AF was introduced into the flow cell using a syringe pump to attain adsorption of BSA-AF onto the pore surface of the PAA layer at a flow rate of 5 μL/min for 90 min.

3. Results and discussion

3-1. Angular reflection and fluorescence spectra measured with the NPWG

Using the optical setup shown in Fig. 2, angular reflection and fluorescence spectra of the PAA/Al bilayer film were measured for adsorption of BSA-AF onto the PAA layer, and typical results are shown in Fig. 3
Fig. 3 Typical reflection (lines with colored squares) and fluorescence (solid lines without marks) spectra of the PAA/Al film measured before (blue lines) and after (red lines) adsorption of 100 nM BSA-AF. Spectra of the PAA/Al film are also shown after rinsing (black lines) with a buffer solution (5 mM phosphate buffer, pH = 4.6). For comparison the reflectivity (green line with squares) and fluorescence (green solid line) are also shown for bare glass for total internal reflection (TIR) after BSA-AF adsorption.
. The dimensions of the PAA/Al film are as follows: thicknesses of the PAA layer and the Al layer are, respectively, 480 nm and 11 nm, and the PAA pore diameter is 16 nm. The PAA pore size is large enough to incorporate BSA-AF into the pores, considering the molecular dimensions of 4 × 4 × 14 nm3 for BSA-AF [25

25. P. G. Squire, P. Moser, and C. T. O’Konski, “The hydrodynamic properties of bovine serum albumin monomer and dimer,” Biochemistry 7(12), 4261–4272 (1968). [CrossRef] [PubMed]

, 26

26. S. J. McClellan and E. I. Franses, “Effect of concentration and denaturation on adsorption and surface tension of bovine serum albumin,” Colloids Surf. B Biointerfaces 28(1), 63–75 (2003). [CrossRef]

].

In the optical waveguide mode, transverse electric (TE) and/or transverse magnetic (TM) modes can be respectively excited by s- and p-polarized light, depending on the experimental conditions [24

24. K. Hotta, A. Yamaguchi, and N. Teramae, “Nanoporous waveguide sensor with optimized nanoarchitectures for highly sensitive label-free biosensing,” ACS Nano 6(2), 1541–1547 (2012).

]. For the configuration of the PAA/Al film, an optical wave guide mode, TE1 can be excited in the PAA layer, and actually a clear waveguide coupling dip is observed at an incident angle of θOWG = 63.58° before adsorption of BSA-AF. Upon adsorption of 100 nM BSA-AF, the coupling dip shifts to the red (θOWG = 63.84°) and the dip position does not change after rinsing the PAA layer with a 5 mM phosphate buffer solution (pH 4.6) for 30 min as seen in Fig. 3. According to the reported Fresnel calculations [27

27. N. Skivesen, R. Horvath, and H. C. Pedersen, “Optimization of metal-clad waveguide sensors,” Sens. Actuators B Chem. 106(2), 668–676 (2005). [CrossRef]

], the shift of the coupling dip can be ascribed to the increase in the RI of the PAA layer and thus, the appearance of the red shift supports the adsorption of BSA-AF to the PAA layer.

In the angular fluorescence spectra shown in Fig. 3, no noticeable response is recognized before adsorption, but a steep increase in the fluorescence response is observed and its maximum is located around θOWG, suggesting that adsorbed BSA-AF is excited by the waveguide mode and emits fluorescence. Similar to the behavior seen in the angular reflection spectra, the fluorescence response does not show noticeable changes after rinsing the PAA layer with a buffer solution, suggesting that the waveguide mode is confined within the PAA layer.

We also measured the fluorescence spectrum in the absence of the waveguide structure, i.e., fluorescence was measured for a bare glass substrate in the total internal reflection (TIR) geometry. In the reflection spectrum measured for the TIR geometry shown in Fig. 3, the critical angle (θC) is observed at 61.50°. However, the fluorescence intensity excited by the evanescent wave formed by the TIR condition (θ > θC) is extremely small. Compared to the TIR geometry, the fluorescence signal is quite high for the PAA/Al film and this enhanced fluorescence can be ascribed to the large adsorbed amount of BSA-AF on the large surface area of the PAA pores and the enhanced field of the waveguide mode excited in the PAA layer.

3-2. Calculation of the field distribution and intensity of the waveguide modes

3-3. Dependence of the fluorescence enhancement on parameters of the PAA layer

Three kinds of PAA/Al films were fabricated with different porosities of the PAA layer by changing the etching time to widen the pores of the PAA layer. First, an Al layer with a thickness of 370 nm was formed on three glass substrates by vapor deposition. Then the PAA layer was formed on the Al layer by anodization at 10 V. Resultant PAA/Al films were treated in an etchant (10 wt% phosphoric acid solution) for 0 s, 320s and 1200s, and these films are respectively designated as Films A, B, and C. The RIs of these films were estimated by the simulation of measured angular reflection spectra with Fresnel calculations and were 1.61, 1.54 and 1.39 for Films A, B and C, respectively. Top and cross-sectional views of SEM images of these films are shown in Fig. 5(a)
Fig. 5 (a) Top and cross-sectional SEM images of the three PAA/Al films used in the fluorescence measurements. (b) (c) Angular reflection (dotted lines) and fluorescence spectra (solid lines) measured for adsorption of BSA-AF onto the three PAA/Al films shown in (a). Scale bars in (a) correspond to 200 nm.
and their structural parameters (thickness of the PAA layer and the PAA pore diameter) estimated from the SEM images are summarized in Table 1

Table 1. Parameters of the PAA Layer in the Films Shown in Fig. 5(c).

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.

Figures 5(b) and 5(c) show angular reflection and fluorescence spectra measured for the three PAA/Al films after adsorption of BSA-AF. For these films, different orders of the waveguide modes are excited depending on the RI of the PAA layer and polarization of incident light, that is, TE1 mode can be excited for Films A and B, and both TE0 and TM1 modes, for Film C. In accordance with the simulated results shown in Fig. 4, the TE0 mode excited in Film C with the lowest nPAA offers the strongest fluorescence intensity among three films as shown in Fig. 5(c). The fluorescence intensity is about 2 times higher than that obtained by the TM1 mode for Film C, and 53 and 4 times higher than that for Films A and B, respectively, excited by the TE1 mode. The high fluorescence intensity is partly due to the strong field enhancement by the TE0 mode and is also due to the larger surface area of Film C compared to the other films. For example, much weaker fluorescence response is recognized for Film A as shown in Fig. 5(b). This weak response is ascribed to the small pore diameter (less than 10 nm) of Film A as shown in the Fig. 5(a) and the small pore diameter prevents adsorption of BAS-AF onto the pore wall surfaces of the PAA layer. Thus, the weak fluorescence response observed for Film A mainly originates from the BSA-AF adsorbed on the top surface of the PAA layer, suggesting that the enhanced fluorescence response observed for Films B and C is caused by the presence of wider nanopores in the PAA waveguiding layer.

As discussed above, the fluorescence enhancement by NPWG can be ascribed to two factors: (a) the large adsorption capacity of the PAA layer and (b) the field enhancement by the waveguide modes. To estimate the contributions of both (a) and (b), figure of merit (FOM) defined by Eq. (1) was introduced by taking a ratio of the fluorescence maximum peak intensity (Fpeak) to the adsorbed amount of BSA-AF (qBSA-AF) per unit surface area of the PAA layer. In the calculation of FOM, 1/cosθpeak factor was incorporated to correct the difference in the incident energy densities of the fluorescence sampling area. In our measurement setups, the diameter of the sample area detected by CCD is ϕ = 0.7 mm and this is smaller than the beam radius of the diode layer (ϕ = 1.0 mm). Thus, the laser energy density transferred into the sampling area is affected by the incident angle of the laser beam (θ) and is approximately proportional to cosθ. The FOM value was estimated for each waveguide mode shown in Fig. 5.

FOM=FpeakqBSA-AF×cosθpeak
(1)

Adsorption of BSA-AF onto the PAA layer causes changes in nPAA, which results in the shift of the wave coupling dip (ΔθOWG) in the angular reflection spectra. Considering the RI of the alumina matrix (nal) that is the part excluding the pore portion of the PAA layer, we can express RI of the PAA pores (npore) and the volume fraction of the pores of the PAA layer (fpore), nPAA as follows [28

28. T. D. Lazzara, K. H. A. Lau, and W. Knoll, “Mounted nanoporous anodic alumina thin films as planar optical waveguides,” J. Nanosci. Nanotechnol. 10(7), 4293–4299 (2010). [CrossRef] [PubMed]

]:
nPAA2=nal2(1-fpore)+npore2fpore
(2)
nPAA2=nal2+nal2fpore(npore2-nal2)βnal2-1/2fpore(npore2-nal2)β
(3)
β=2npore2/nal2+1
(4)
where Eq. (2) expresses the dielectric constant of the PAA layer (εPAA = nPAA2) in the direction normal to the film surface, while Eq. (3) is the value in the direction parallel to the film surface. RI of the PAA pores covered with some amount of BSA-AF can be expressed as a function of RI of BSA-AF, RI of water, and the volume fraction of adsorbed BSA [24

24. K. Hotta, A. Yamaguchi, and N. Teramae, “Nanoporous waveguide sensor with optimized nanoarchitectures for highly sensitive label-free biosensing,” ACS Nano 6(2), 1541–1547 (2012).

]. Using the relationships described above, qBSA-AF can be estimated by fitting measured angular spectra with simulated results obtained by Fresnel calculations. In the Fresnel calculations, thickness (dPAA) and fpore of the PAA layer were estimated from the SEM measurements (Table 1). RI values of the densely packed BSA-AF layer and the buffer solution were set as 1.47 [24

24. K. Hotta, A. Yamaguchi, and N. Teramae, “Nanoporous waveguide sensor with optimized nanoarchitectures for highly sensitive label-free biosensing,” ACS Nano 6(2), 1541–1547 (2012).

, 26

26. S. J. McClellan and E. I. Franses, “Effect of concentration and denaturation on adsorption and surface tension of bovine serum albumin,” Colloids Surf. B Biointerfaces 28(1), 63–75 (2003). [CrossRef]

] and 1.35, respectively. The density of BSA-AF was assumed to be 0.49 g/cm3 by considering its molecular weight (67000 Dalton) [25

25. P. G. Squire, P. Moser, and C. T. O’Konski, “The hydrodynamic properties of bovine serum albumin monomer and dimer,” Biochemistry 7(12), 4261–4272 (1968). [CrossRef] [PubMed]

].

In the simulation for Film A, qBSA-AF was estimated on the assumption that BSA-AF adsorbed on the top surface of the PAA layer to form a monolayer of BSA-AF with thickness of 7 nm [29

29. K. H. A. Lau, L. S. Tan, K. Tamada, M. S. Sander, and W. Knoll, “Highly sensitive detection of process occurring inside nanoporous anodic alumina templates: a waveguide optical study,” J. Phys. Chem. B 108(30), 10812–10818 (2004). [CrossRef]

]. For Films B and C, adsorption of BSA-AF was assumed to occur homogeneously on their top surfaces and in the cylindrical nanopores of the PAA layer. Calculated results of qBSA-AF and FOM are summarized in Table 2

Table 2. FOM and the Values Used to Obtain FOM Values (n = 3)

table-icon
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with the experimental values (Fpeak and ΔθOWG) used for the calculations. For each film, field distribution was calculated according to the reported procedures [20

20. W. N. Hansen, “Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium,” J. Opt. Soc. Am. 58(3), 380–390 (1968). [CrossRef]

] and the averaged field intensity within the PAA layer was also calculated. This averaged field intensity corresponds to the field enhancement factor (EF) and EF values are also listed in Table 2.

As seen in Table 2, both the large adsorption capacity of the PAA layer of the films and the field enhancement by the waveguide modes are important for NPWG to enhance the fluorescence responses. The estimated qBSA-AF values for Film C by TE0 and TM1 modes are comparable with each other within the errors, and the qBSA-AF value is 6 and 1.1 times larger than that for Films A and B, respectively. FOM calculated for the TE0 mode of Film C is ca. 2 times higher than that for the TM1 mode and ca. 9 and ca. 3 times higher than that for the TE1 modes of Films A and B, respectively. Although the calculated FOM values reasonably agree with the order of the EF values, FOM is not directly proportional to the EF values. This can be ascribed to a coupling efficiency of the field intensity with fluorophores, scattering of light at the PAA pores, and the orientation of BSA-AF at the PAA pore surface. Finally, it should be noted that more than 140-fold enhanced fluorescence (Table 2, averaged peak fluorescence intensity is 41900 counts) is achieved for Film C excited by the TE0 mode, compared to the fluorescence intensity measured under the TIR condition (Fig. 3, averaged fluorescence intensity is 288 counts).

4. Conclusion

We investigated fluorescence behavior from a fluorophore-labeled BSA adsorbed on the NPWG film comprised of a PAA layer on an Al layer. Based on the measurements of angular reflection and angular fluorescence spectra for adsorption of BSA-AF on the PAA layer, we found that the waveguide modes excited in the PAA layer can be used for the selective excitation of fluorophores in the PAA nanopores of the NPWG film. The fluorescence intensity for the NPWG was much larger than that measured for the substrate without the waveguide structure (TIR configuration). From simulation results of the field distributions within the NPWG film, we expected an enhanced field distribution for the TE0 mode excited in the PAA layer with low RI. Quantitative analysis based on the Fresnel calculations revealed that the enhanced fluorescence response for the NPWG film was achieved by both the increase in the adsorption capacity due to the high surface area of the PAA pores and the field enhancement in the PAA layer by the waveguide modes.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research (No. 21685009 and No. 22225003) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References and links

1.

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008). [CrossRef]

2.

J. R. Lakowicz, K. Ray, M. Chowdhury, H. Szmacinski, Y. Fu, J. Zhang, and K. Nowaczyk, “Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy,” Analyst (Lond.) 133(10), 1308–1346 (2008). [CrossRef] [PubMed]

3.

W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt. 45(4), 661–699 (1998). [CrossRef]

4.

F. D. Stefani, K. Vasilev, N. Bocchio, F. Gaul, A. Pomozzi, and M. Kreiter, “Photonic mode density effects on single-molecule fluorescence blinking,” New J. Phys. 9(2), 21 (2007). [CrossRef]

5.

J. Zhang and J. R. Lakowicz, “Metal-enhanced fluorescence of an organic fluorophore using gold particles,” Opt. Express 15(5), 2598–2606 (2007). [CrossRef] [PubMed]

6.

Y. Chen, K. Munechika, and D. S. Ginger, “Dependence of fluorescence intensity on the spectral overlap between fluorophores and plasmon resonant single silver nanoparticles,” Nano Lett. 7(3), 690–696 (2007). [CrossRef] [PubMed]

7.

R. Bardhan, N. K. Grady, J. R. Cole, A. Joshi, and N. J. Halas, “Fluorescence enhancement by Au nanostructures: nanoshells and nanorods,” ACS Nano 3(3), 744–752 (2009). [CrossRef] [PubMed]

8.

Y. Fu, J. Zhang, and J. R. Lakowicz, “Plasmon-enhanced fluorescence from single fluorophores end-linked to gold nanorods,” J. Am. Chem. Soc. 132(16), 5540–5541 (2010). [CrossRef] [PubMed]

9.

S. Wedge and W. L. Barnes, “Surface plasmon-polariton mediated light emission through thin metal films,” Opt. Express 12(16), 3673–3685 (2004). [CrossRef] [PubMed]

10.

S. Fang, H. J. Lee, A. W. Wark, H. M. Kim, and R. M. Corn, “Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy,” Anal. Chem. 77(20), 6528–6534 (2005). [CrossRef] [PubMed]

11.

S.-H. Guo, J. J. Heetderks, H.-C. Kan, and R. J. Phaneuf, “Enhanced fluorescence and near-field intensity for Ag nanowire/nanocolumn arrays: evidence for the role of surface plasmon standing waves,” Opt. Express 16(22), 18417–18425 (2008). [CrossRef] [PubMed]

12.

J. P. Hoogenboom, G. Sanchez-Mosteiro, G. Colas des Francs, D. Heinis, G. Legay, A. Dereux, and N. F. van Hulst, “The single molecule probe: Nanoscale vectorial mapping of photonic mode density in a metal nanocavity,” Nano Lett. 9(3), 1189–1195 (2009). [CrossRef] [PubMed]

13.

P.-F. Guo, S. Wu, Q.-J. Ren, J. Lu, Z. Chen, S.-J. Xiao, and Y.-Y. Zhu, “Fluorescence enhancement by surface plasmon polaritons on metallic nanohole arrays,” J. Phys. Chem. Lett. 1(1), 315–318 (2010). [CrossRef]

14.

Y.-J. Hung, I. I. Smolyaninov, C. C. Davis, and H. C. Wu, “Fluorescence enhancement by surface gratings,” Opt. Express 14(22), 10825–10830 (2006). [CrossRef] [PubMed]

15.

Y. Jiang, H.-Y. Wang, H. Wang, B.-R. Gao, Y.- Hao, Y. Jin, Q.-D. Chen, and H.-B. Sun, “Surface plasmon enhanced fluorescence of dye molecules on metal grating films,” J. Phys. Chem. C 115(25), 12636–12642 (2011). [CrossRef]

16.

N. Ganesh, P. C. Mathias, W. Zhang, and B. T. Cunningham, “Distance dependence of fluorescence enhancement from photonic crystal surface,” J. Appl. Phys. 103(8), 083104 (2008). [CrossRef]

17.

A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express 14(8), 3512–3527 (2006). [CrossRef] [PubMed]

18.

N. Ganesh, I. D. Block, P. C. Mathias, W. Zhang, E. Chow, V. Malyarchuk, and B. T. Cunningham, “Leaky-mode assisted fluorescence extraction: application to fluorescence enhancement biosensors,” Opt. Express 16(26), 21626–21640 (2008). [CrossRef] [PubMed]

19.

C. J. Huang, J. Dostalek, and W. Knoll, “Long range surface plasmon and hydrogel optical waveguide field-enhanced fluorescence biosensor with 3D hydrogel binding matrix: On the role of diffusion mass transfer,” Biosens. Bioelectron. 26(4), 1425–1431 (2010). [CrossRef] [PubMed]

20.

W. N. Hansen, “Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium,” J. Opt. Soc. Am. 58(3), 380–390 (1968). [CrossRef]

21.

S. Fukuzaki, H. Urano, and K. Nagata, “Adsorption of bovine serum albumin onto metal oxide surfaces,” J. Ferment. Bioeng. 81(2), 163–167 (1996). [CrossRef]

22.

A. Yamaguchi, K. Hotta, and N. Teramae, “Optical waveguide sensor based on a porous anodic alumina/aluminum multilayer film,” Anal. Chem. 81(1), 105–111 (2009). [CrossRef] [PubMed]

23.

K. Hotta, A. Yamaguchi, and N. Teramae, “Properties of a metal clad waveguide sensor based on a nanoporous-metal-oxide/metal multilayer film,” Anal. Chem. 82(14), 6066–6073 (2010). [CrossRef] [PubMed]

24.

K. Hotta, A. Yamaguchi, and N. Teramae, “Nanoporous waveguide sensor with optimized nanoarchitectures for highly sensitive label-free biosensing,” ACS Nano 6(2), 1541–1547 (2012).

25.

P. G. Squire, P. Moser, and C. T. O’Konski, “The hydrodynamic properties of bovine serum albumin monomer and dimer,” Biochemistry 7(12), 4261–4272 (1968). [CrossRef] [PubMed]

26.

S. J. McClellan and E. I. Franses, “Effect of concentration and denaturation on adsorption and surface tension of bovine serum albumin,” Colloids Surf. B Biointerfaces 28(1), 63–75 (2003). [CrossRef]

27.

N. Skivesen, R. Horvath, and H. C. Pedersen, “Optimization of metal-clad waveguide sensors,” Sens. Actuators B Chem. 106(2), 668–676 (2005). [CrossRef]

28.

T. D. Lazzara, K. H. A. Lau, and W. Knoll, “Mounted nanoporous anodic alumina thin films as planar optical waveguides,” J. Nanosci. Nanotechnol. 10(7), 4293–4299 (2010). [CrossRef] [PubMed]

29.

K. H. A. Lau, L. S. Tan, K. Tamada, M. S. Sander, and W. Knoll, “Highly sensitive detection of process occurring inside nanoporous anodic alumina templates: a waveguide optical study,” J. Phys. Chem. B 108(30), 10812–10818 (2004). [CrossRef]

OCIS Codes
(230.7370) Optical devices : Waveguides
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(160.4236) Materials : Nanomaterials
(280.4788) Remote sensing and sensors : Optical sensing and sensors

ToC Category:
Spectroscopy

History
Original Manuscript: February 10, 2012
Revised Manuscript: March 18, 2012
Manuscript Accepted: March 19, 2012
Published: May 23, 2012

Citation
Yong Fan, Kazuhiro Hotta, Akira Yamaguchi, and Norio Teramae, "Enhanced fluorescence in a nanoporous waveguide and its quantitative analysis," Opt. Express 20, 12850-12859 (2012)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-20-12-12850


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References

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  2. J. R. Lakowicz, K. Ray, M. Chowdhury, H. Szmacinski, Y. Fu, J. Zhang, and K. Nowaczyk, “Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy,” Analyst (Lond.)133(10), 1308–1346 (2008). [CrossRef] [PubMed]
  3. W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt.45(4), 661–699 (1998). [CrossRef]
  4. F. D. Stefani, K. Vasilev, N. Bocchio, F. Gaul, A. Pomozzi, and M. Kreiter, “Photonic mode density effects on single-molecule fluorescence blinking,” New J. Phys.9(2), 21 (2007). [CrossRef]
  5. J. Zhang and J. R. Lakowicz, “Metal-enhanced fluorescence of an organic fluorophore using gold particles,” Opt. Express15(5), 2598–2606 (2007). [CrossRef] [PubMed]
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  7. R. Bardhan, N. K. Grady, J. R. Cole, A. Joshi, and N. J. Halas, “Fluorescence enhancement by Au nanostructures: nanoshells and nanorods,” ACS Nano3(3), 744–752 (2009). [CrossRef] [PubMed]
  8. Y. Fu, J. Zhang, and J. R. Lakowicz, “Plasmon-enhanced fluorescence from single fluorophores end-linked to gold nanorods,” J. Am. Chem. Soc.132(16), 5540–5541 (2010). [CrossRef] [PubMed]
  9. S. Wedge and W. L. Barnes, “Surface plasmon-polariton mediated light emission through thin metal films,” Opt. Express12(16), 3673–3685 (2004). [CrossRef] [PubMed]
  10. S. Fang, H. J. Lee, A. W. Wark, H. M. Kim, and R. M. Corn, “Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy,” Anal. Chem.77(20), 6528–6534 (2005). [CrossRef] [PubMed]
  11. S.-H. Guo, J. J. Heetderks, H.-C. Kan, and R. J. Phaneuf, “Enhanced fluorescence and near-field intensity for Ag nanowire/nanocolumn arrays: evidence for the role of surface plasmon standing waves,” Opt. Express16(22), 18417–18425 (2008). [CrossRef] [PubMed]
  12. J. P. Hoogenboom, G. Sanchez-Mosteiro, G. Colas des Francs, D. Heinis, G. Legay, A. Dereux, and N. F. van Hulst, “The single molecule probe: Nanoscale vectorial mapping of photonic mode density in a metal nanocavity,” Nano Lett.9(3), 1189–1195 (2009). [CrossRef] [PubMed]
  13. P.-F. Guo, S. Wu, Q.-J. Ren, J. Lu, Z. Chen, S.-J. Xiao, and Y.-Y. Zhu, “Fluorescence enhancement by surface plasmon polaritons on metallic nanohole arrays,” J. Phys. Chem. Lett.1(1), 315–318 (2010). [CrossRef]
  14. Y.-J. Hung, I. I. Smolyaninov, C. C. Davis, and H. C. Wu, “Fluorescence enhancement by surface gratings,” Opt. Express14(22), 10825–10830 (2006). [CrossRef] [PubMed]
  15. Y. Jiang, H.-Y. Wang, H. Wang, B.-R. Gao, Y.- Hao, Y. Jin, Q.-D. Chen, and H.-B. Sun, “Surface plasmon enhanced fluorescence of dye molecules on metal grating films,” J. Phys. Chem. C115(25), 12636–12642 (2011). [CrossRef]
  16. N. Ganesh, P. C. Mathias, W. Zhang, and B. T. Cunningham, “Distance dependence of fluorescence enhancement from photonic crystal surface,” J. Appl. Phys.103(8), 083104 (2008). [CrossRef]
  17. A. Minardo, R. Bernini, F. Mottola, and L. Zeni, “Optimization of metal-clad waveguides for sensitive fluorescence detection,” Opt. Express14(8), 3512–3527 (2006). [CrossRef] [PubMed]
  18. N. Ganesh, I. D. Block, P. C. Mathias, W. Zhang, E. Chow, V. Malyarchuk, and B. T. Cunningham, “Leaky-mode assisted fluorescence extraction: application to fluorescence enhancement biosensors,” Opt. Express16(26), 21626–21640 (2008). [CrossRef] [PubMed]
  19. C. J. Huang, J. Dostalek, and W. Knoll, “Long range surface plasmon and hydrogel optical waveguide field-enhanced fluorescence biosensor with 3D hydrogel binding matrix: On the role of diffusion mass transfer,” Biosens. Bioelectron.26(4), 1425–1431 (2010). [CrossRef] [PubMed]
  20. W. N. Hansen, “Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium,” J. Opt. Soc. Am.58(3), 380–390 (1968). [CrossRef]
  21. S. Fukuzaki, H. Urano, and K. Nagata, “Adsorption of bovine serum albumin onto metal oxide surfaces,” J. Ferment. Bioeng.81(2), 163–167 (1996). [CrossRef]
  22. A. Yamaguchi, K. Hotta, and N. Teramae, “Optical waveguide sensor based on a porous anodic alumina/aluminum multilayer film,” Anal. Chem.81(1), 105–111 (2009). [CrossRef] [PubMed]
  23. K. Hotta, A. Yamaguchi, and N. Teramae, “Properties of a metal clad waveguide sensor based on a nanoporous-metal-oxide/metal multilayer film,” Anal. Chem.82(14), 6066–6073 (2010). [CrossRef] [PubMed]
  24. K. Hotta, A. Yamaguchi, and N. Teramae, “Nanoporous waveguide sensor with optimized nanoarchitectures for highly sensitive label-free biosensing,” ACS Nano6(2), 1541–1547 (2012).
  25. P. G. Squire, P. Moser, and C. T. O’Konski, “The hydrodynamic properties of bovine serum albumin monomer and dimer,” Biochemistry7(12), 4261–4272 (1968). [CrossRef] [PubMed]
  26. S. J. McClellan and E. I. Franses, “Effect of concentration and denaturation on adsorption and surface tension of bovine serum albumin,” Colloids Surf. B Biointerfaces28(1), 63–75 (2003). [CrossRef]
  27. N. Skivesen, R. Horvath, and H. C. Pedersen, “Optimization of metal-clad waveguide sensors,” Sens. Actuators B Chem.106(2), 668–676 (2005). [CrossRef]
  28. T. D. Lazzara, K. H. A. Lau, and W. Knoll, “Mounted nanoporous anodic alumina thin films as planar optical waveguides,” J. Nanosci. Nanotechnol.10(7), 4293–4299 (2010). [CrossRef] [PubMed]
  29. K. H. A. Lau, L. S. Tan, K. Tamada, M. S. Sander, and W. Knoll, “Highly sensitive detection of process occurring inside nanoporous anodic alumina templates: a waveguide optical study,” J. Phys. Chem. B108(30), 10812–10818 (2004). [CrossRef]

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