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Cientificas), Isaac Newton 8, 28760 Tres Cantos, Madrid, Spain ... The characteristics of a novel magneto-optic surface-plasmon-resonance (MOSPR) sensor ...
April 15, 2006 / Vol. 31, No. 8 / OPTICS LETTERS

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Highly sensitive detection of biomolecules with the magneto-optic surface-plasmon-resonance sensor B. Sepúlveda, A. Calle, L. M. Lechuga, and G. Armelles Instituto de Microelectrónica de Madrid (Centro Nacional de Microelectrónica—Consejo Superior de Investigaciones Cientificas), Isaac Newton 8, 28760 Tres Cantos, Madrid, Spain Received October 21, 2005; revised January 12, 2006; accepted January 18, 2006; posted January 27, 2006 (Doc. ID 65527) The characteristics of a novel magneto-optic surface-plasmon-resonance (MOSPR) sensor and its use for the detection of biomolecules are presented. This physical transduction principle is based on the combination of the magneto-optic activity of magnetic materials and a surface-plasmon resonance of metallic layers. Such a combination can produce a sharp enhancement of the magneto-optic effects that strongly depends on the optical properties of the surrounding medium, allowing its use for biosensing applications. Experimental characterizations of the MOSPR sensor have shown an increase in the limit of detection by a factor of 3 in changes of refractive index and in the adsorption of biomolecules compared with standard sensors. Optimization of the metallic layers and the experimental setup could result in an improvement of the limit of detection by as much as 1 order of magnitude. © 2006 Optical Society of America OCIS codes: 230.0230, 2303810, 2406680, 130.6010.

Since the first application of surface-plasmon resonance (SPR) as a biosensor in 1982,1,2 this analytical technique has experienced a wide development. SPR is a charge density oscillation that generates highly confined electromagnetic fields at the interface of a metal and a dielectric. The excitation condition of the SPR strongly depends on the refractive index of the dielectric medium; this dependence is the operating principle of detection of the SPR biosensors. The sensitivity and limits of detection of the various SPR sensors show variations that depend on the method used to excite the surface plasmon3–6 but can be set to the order of 10−5 in bulk refractive-index changes, which corresponds to a limit of detection of 1 – 5 pg mm−2 of biomolecules adsorbed at the sensor surface. However, such resolution is not enough for the direct detection of low concentrations of small molecules, for which detection limits of ⬃0.1 pg mm−2 or lower are necessary. Recently several configurations to improve such limits of detection, i.e., phasesensitive SPR based on a Mach–Zehnder configuration,7 differential ellipsometric SPR,8 optical heterodyne SPR,9 and SPR with external magneto-optic (MO) polarization modulation,10 were described. Within this context, we propose a novel magnetooptical SPR11 sensor based on a combination of the MO activity of the magnetic materials and SPR. This combination produces a great enhancement of MO Kerr effects of p-polarized light when the resonant condition is satisfied.12,13 MO Kerr effects depend on the direction of the magnetization of the magnetic layer with respect to the direction of propagation of the incident light. If the magnetization is perpendicular to the plane of the layer (polar configuration), the MO activity induces rotation and a change in ellipticity of the polarization plane of the reflected light. Meanwhile, if the magnetization is perpendicular to the propagation plane of the light (transversal configuration), the MO effect produces a relative change 0146-9592/06/081085-3/$15.00

of the reflectivity 共Rpp兲 of the p-polarized light: ⌬Rpp/Rpp = Rpp共M兲 − Rpp共0兲/Rpp共0兲,

共1兲

where Rpp共M兲 and Rpp共0兲 represent the reflectivities with and without magnetization, respectively. When the surface plasmon is excited, Rpp is largely reduced while the MO components (e.g., ⌬Rpp) are maintained or even increased, producing a sharp enhancement of the MO effect in the reflected light. As the condition of excitation of SPR strongly depends on the refractive index of the dielectric medium 共nd兲, the MO measurements will be highly sensitive to the changes of nd, permitting the measurements’ use for optical biosensing. The simplest combination of SPR and MO activity can be found by use of ferromagnetic metals. However, the combination of ferromagnetic metals with the metals typically used in plasmonic applications (Ag and Au) is more sensitive and interesting for biosensing applications. As an example, we analyze the theoretical MO effects in the transversal configuration of the magnetization 共⌬Rpp / Rpp兲 for a Co layer of 20 nm as a function of the angle of incidence, compared to the MO effects of a Co/ Au multilayer composed of 10 nm of Co and 29.4 nm of Au. In these calculations we assume a Kretschmann configuration to excite the SPR, glass as the incident medium 共ni = 1.5200兲, and water as the outer medium 共nd = 1.3323兲. The dielectric and MO constants of Co are ⑀xxCo = −11.43+ i18.15 and ⑀xzCo = −0.65+ i0.0005, respectively, at a wavelength of 632 nm, whereas the dielectric constant of Au is ⑀xxAu = −11.00+ i1.98.13,14 As can be observed from Fig. 1(a), the MO effects are extremely strong and are closely localized at the condition of excitation of SPR, which is an ⬃73° incidence angle for water. To quantify and compare the sensitivity of the MO effects we analyze the variation of the acquired measurement 共S兲 when nd changes: © 2006 Optical Society of America

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Fig. 1. (a) Comparison of ⌬Rpp / Rpp for a Co layer of 20 nm, a Co/ Au multilayer that comprises 10 nm of Co and 29.4 nm of Au, and a Cr/ Co/ Cr/ Au multilayer with 2 and 3 nm of Cr, 7.5 nm of Co, and 23 nm of Au. (b) Sensitivity of ⌬Rpp / Rpp in each configuration.

␩ = ⳵ S/⳵ nd = 共⳵ S/⳵ A兲共⳵ A/⳵ nd兲,

共2兲

where S could be Rpp (SPR standard measurement); ⌬Rpp / Rpp is the Kerr rotation or ellipticity; and A is the incidence angle of the light. Although the MO effects of the single 20 nm Co layer are stronger than the effects of the Co/ Au multilayer, Fig. 1(b) shows that ␩ of the latter is approximately four times greater. From Eq. (2) we can appreciate that ␩ depends on two factors, the slope of the measured curve as a function of the angle of incidence 共A兲 and the angular displacement of such a curve when nd changes. In the Co/ Au multilayer, both the slope and the angular displacement of the MO effects are greater than in 20 nm Co layer, which explains the enhancement of ␩. Such sensitivity is also highly dependent on the thickness of the metallic layers. In both configurations we chose the metallic thicknesses to maximize the sensitivity. In the polar configuration, the same behavior is found in the Kerr rotation or ellipticity. For the experimental development of the MOSPR sensor we chose the combination of Co and Au layers. The metallic layers are deposited by electron-beam evaporation onto glass slides. In these conditions the Co layer is polycrystalline and has in-plane magnetization (saturation magnetic field, 50 Oe). As a consequence, we use the transversal configuration of the magnetization, measuring ⌬Rpp / Rpp. These measurements require a change in the state of the magnetization, as can be deduced from Eq. (1), which was made with rotating magnets. In our experimental setup the incident light of a He– Ne laser is p polarized and prism coupled to excite SPR, and the reflected light is collected with a photodiode. A flow cell and a peristaltic pump are used to control the various solutions. Finally, the sensor is mounted on a rotation stage for angular interrogation. To improve the adhesion and stability of the Co/ Au multilayer, two thin Cr layers are introduced, the first one over the glass and the second between the Co and the Au layers. The strong absorption of Cr layers forces reduction of the thicknesses of the Au and Co layers to pro-

duce enhancement of the MO effects. In this configuration the most sensitive MO effects have been obtained for multilayers of 2 nm of Cr, 7.5 nm of Co, 3 nm of Cr, and 23 nm of Au. Such MO effects are represented in Fig. 2, together with the theoretical calculations, assuming that the dielectric constant of Cr at 632 nm is ⑀xxCr = −6.88+ i29.30.14 As can be observed, there is very good agreement between the experimental results and the theoretical calculation. Owing to the different natures of the MO measurements of the MOSPR sensor and the Rpp measurements of the SPR sensor, a comparison of their sensitivities must take into account the signal-to-noiseratio of the experimental measurements. First we define the minimum detectable signal 共Smin兲 as five times the root-mean-square deviation of the experimental signal acquired during 300 s when 1 point/s is represented. Accordingly, the experimental sensitivity 共␩E兲 of the sensor to changes in refractive index can be represented as ␩ normalized by Smin:

␩E = ␩/Smin .

共3兲

Using this expression, we calculate the experimental limit of detection of the sensor in changes of refractive index 共nEmin兲 as

␩Emin = 1/␩E .

共4兲

We perform an evaluation and a comparison of the sensitivities and the limits of detection of the MOSPR and SPR devices by flowing solutions of different refractive indices and measuring, in real time, the variations of ⌬Rpp / Rpp and Rpp, respectively, at a fixed angle of incidence, using the same experimental setup. Such an angle of incidence was chosen to maximize the slope of the ⌬Rpp / Rpp (MOSPR) or the Rpp (SPR) curves. In Fig. 3 we represent the MO response of the MOSPR sensor to changes of nd normalized by Smin, which is 1 ⫻ 10−3 in our experiments. These results are compared with the evaluation of ␩E for a standard SPR sensor whose sensing surface is formed by 2 nm Cr and 45 nm Au. In this case the minimum detectable Rpp is 5 ⫻ 10−4. A comparison of the two measurements shows that ␩E of the MOSPR sensor is approximately three times greater than the

Fig. 2. Experimental and theoretical curves of ⌬Rpp / Rpp of the Cr/ Co/ Cr/ Au multilayer as a function of the angle of incidence.

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In conclusion, we have presented a new MOSPR biosensor that improves the experimental sensitivity of standard SPR sensors threefold. The simplicity of the experimental setup, its immunity to fluctuations of the light source, and its compatibility with the well-known thiol immobilization chemistry of gold add interest to this novel concept of a biosensor. Optimization of the metallic layers and of the experimental setup could result in an improvement in the limit of detection of as much as 1 order of magnitude with respect to conventional SPR sensors.

Fig. 3. Comparison of the experimental normalized signals of the MOSPR and the SPR sensors that are due to refractive-index changes, and evaluation of their experimental sensitivities. Inset, Normalized signal of the detection of the physical adsorption of bovine serum albumin proteins.

sensitivity of the SPR sensor. The ␩E value obtained for the Cr/ Co/ Cr/ Au multilayer corresponds to a sensitivity 共␩兲 of the MO measurements of 191, very close to the theoretical result predicted in Fig. 1(b). Finally, using the Eq. (4) and the ␩E values obtained from Fig. 3, we can set the limits of detection in changes of refractive index to 5 ⫻ 10−6 and 1.5⫻ 10−5 for the MOSPR and SPR sensors, respectively. Once ␩E is evaluated, we test the response of the MOSPR device for the adsorption of bovine serum albumin proteins, flowing a 10 ␮g / ml solution in phosphate buffer saline. As the inset of Fig. 3 illustrates, the normalized signal of the MOSPR sensor is again three times higher than the normalized signal of the conventional SPR sensor. Our preliminary results have proved the enhancement of the sensitivity of the MOSPR sensor with respect to the SPR sensor. However, this enhancement can go further with optimization of the deposition processes and a more accurate control of the thickness of the metallic layers. Such optimizations will improve the stability of the MO multilayer in contact with the liquid flow, permitting reduction of the thickness of the Cr layers, which is responsible for the drastic reduction of the sensitivity of the MO measurements [reduction by a factor of 2.8, as Fig. 1(b) shows]. In addition, improvement of the signalto-noise-ratio of the experimental setup by reducing the fluctuations of the rotation frequency of the magnets and the mechanical vibrations will contribute to a decrease nEmin of the MOSPR of as much as 1 order of magnitude compared with SPR.

This work was supported by the Ministerio de Educacion y Ciencia (Spain), Project MAT2002-04484C03-01, and the PHOREMOST European Excellence Network. B. Sepúlveda ([email protected]) acknowledges financial support from the Consejo Superior de Investigaciones Cientificas (Spain). References 1. C. Nylander, B. Liedberg, and T. Lind, Sens. Actuators 3, 79 (1982). 2. B. Liedberg, C. Nylander, and I. Lundström, Sens. Actuators 4, 299 (1983). 3. J. Homola, S. S. Yee, and G. Gauglitz, Sens. Actuators B 54, 3 (1999). 4. A. A. Kolomenskii, P. D. Gershon, and H. A. Schuessler, Appl. Opt. 36, 6539 (1997). 5. T. Chinowsky, L. Jung, and S. Yee, Sens. Actuators B 54, 89 (1999). 6. L. M. Lechuga, in Biosensors and Modern Biospecific Analytical Techniques, L. Gorton, ed., Comprehensive Analytical Chemistry Series, XLIV (Elsevier, 2005), Chap. 5. 7. S. Y. Wu, H. P. Ho, W. C. Law, C. Liu, and S. K. Kong, Opt. Lett. 29, 2378 (2004). 8. I. R. Hooper and J. R. Sambles, Appl. Phys. Lett. 85, 3017 (2004). 9. W. C. Kuo, C. Chou, and H. T. Wu, Opt. Lett. 28, 1329 (2003). 10. J. Guo, Z. Zhu, and W. Deng, Appl. Opt. 38, 6550 (1999). 11. B. Sepúlveda, G. Armelles, L. M. Lechuga, and A. Calle, “Device and method for detecting changes in the refractive index of a dielectric medium,” Spain Patent Application P200401037–PCT/EP2005/006273 (June 11, 2004). 12. P. E. Ferguson, O. M. Stafsudd, and R. F. Wallis, Physica B ⫹ C 89B, 91 (1977). 13. C. Hermann, V. A. Kosobukin, G. Lampel, J. Peretti, V. I. Safarov, and P. Bertrand, Phys. Rev. B 64, 235422 (2001). 14. J. H. Weaver, C. Krafka, D. W. Lynch, and E. E. Kock, in Physics Data: Optical Properties of Metals, H. Behrens, ed. (Fachinformationszentrum Karlsruhe, 1981), p. 45.