BioSystems 98 (2009) 5155

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BioSystems
journal homepage: www.elsevier.com/locate/biosystems

Variable wavelength surface plasmon resonance (SPR) in biosensing

Nyeon-Sik Eum a , Do-Eok Kim b , Se-Hyuk Yeom b , Byoung-Ho Kang b , Kyu-Jin Kim b , Chang-Sub Park c , Shin-Won Kang b,
a

Department of Agriculture and Biological Engineering, Purdue University, 225 South University Street, West Lafayette, IN 47907, USA School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu 702-701, South Korea c Department of Sensor and Display Engineering, Kyungpook National University, Daegu 702-701, South Korea
b

a r t i c l e

i n f o

a b s t r a c t
In this study, we fabricated a novel variable wavelength surface plasmon resonance (SPR) sensor, which detects resonance conditions such as a maximum attenuation wavelength, measuring change of microscopic refractive index. Such a change was measured to detect a salmonella antigenantibody reaction and a penicillinasepenicillin reaction. Our experiments were performed after immobilizing a salmonella antibody on the sensor chip. We measured the shift in resonant wavelength during the antigenantibody reaction for 30 min by injecting 5 107 cells/ml concentration of salmonella antigen solution into the sample chamber. Also, after immobilizing penicillinase on the sensor chip, we measured the shift in resonant wavelength during the reaction. Penicillin solution at 10 mM was injected into the sample chamber. The shift of resonant wavelength for each experiment was measured using a white light source, multimode optical ber, a part of sensor chip and an optical spectrum analyzer. As a result, the resonant wavelength shifted about 0.26 nm/min owing to the salmonella antibodyantigen reaction. Thus, we could detect the change in wavelength (0.8 nm/min) through the interaction of penicillin and penicillinase for 15 min using variable wavelength SPR sensor. 2009 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 12 March 2009 Received in revised form 8 May 2009 Accepted 17 May 2009 Keywords: Variable wavelength Surface plasmon resonance Salmonella Penicillin

1. Introduction The SPR has become widely used in the elds of chemistry and biochemistry to characterize a biological surface (Hoa et al., 2007) and to monitor binding events (Mao and Brody, 2007; Rella et al., 2004). The SPR sensor has many merits such as high sensitivity, light weight, low cost, and linear characteristics. Thus, it has been applied in chemical sensors and biosensors. Considering the research and development in bioengineering in recent years, interest in the measurement of various biomaterials is increasing. In addition, research on overcoming the detecting limit is actively progressing and improving measurement systems as well as the methods for detecting measurement technologies. The methods of detecting biomaterials using optics with high sensitivity are attenuated total reection (ATR) (Soldatkin et al., 2003) using a high-refraction index prism, a ber-optic optode (Rosenzweig and Kopelman, 1996) and thin-lm optical waveguide devices. These methods have a relatively high accuracy, but they also have disadvantages, like as requiring pretreatment, long analysis time, and difculties in manufacturing processes and applications.

Currently, sensors using the SPR are often used in antigenantibody reactions (Tang et al., 2007; Ayela et al., 2007), protein and DNA detection (Yang et al., 2007). Most of the sensors using the SPR phenomenon are Kretschmann-type (Ritchie, 1957; Eum et al., 2003) SPR sensors, and in some cases, a few researchers use an SPR sensor with optical waveguides (Dostalek et al., 2001) and optical bers. In this study, we fabricated a variable wavelength SPR sensor using a white light source (4001800 nm) and multimode optical ber, and observed the movement of resonant wavelength using an optical spectrum analyzer (OSA). The fabricated SPR sensor, which has a variable wavelength, has a wider sensing area than SPR sensors using monochromatic light source. It could measure the shift in resonant wavelength for a smaller change in refractive index of sample than that of a Kretschmann-type SPR which has been normally used until now. In addition, we conrmed the feasibility of a biosensor by measuring the movement of resonant wavelength on the basis of the antibodyantigen reaction of salmonella and the interaction between penicillinase and penicillin. 2. Theory As has been known for several decades, the SPR phenomenon is a surface electromagnetic wave that propagates along the interface between a metal and a dielectric material. A model predicting

Corresponding author. Tel.: +82 53 950 6829; fax: +82 53 950 7932. E-mail address: swkang@knu.ac.kr (S.-W. Kang). 0303-2647/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.biosystems.2009.05.008

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resonance conditions can be obtained using the plane-wave Fresnel reection equations for a structure containing multiple planar layers. Surface plasmons (SPs) are excited by incident laser beams, causing resonance with evanescent waves at a certain resonance angle or resonance wavelength. The SPs are resonated with evanescent waves and decay exponentially away from the phase boundary with a penetration depth on the order of 200 nm (Brockman and Nelson, 2000). This is the so-called attenuated total reectance (ATR) method proposed by Kretschmann. The propagation constant of surface plasmon waves (SPWs) can be expressed as kSPW = k0 m n2 s m + n2 s (1)

where k0 is the propagation constant of incident light in the free space (k0 = 2 / 0 , 0 being the free space wavelength), m is the direct constant of gold, and ns is the refractive index of the solution. The basic principle of the SPR sensor is due to the complex refraction index of a thin metal lm. Thus, resonance occurs according to the changes in dielectric constant, incidence angle, and incident wavelength. In particular, in the case of the variable wavelength SPR sensor, resonance occurs at a xed incidence angle when the wave vector of incident light and that of a surface plasmon become equal, that is, while satisfying the phase-matching condition kev = ksp . After xing the incidence angle under this resonance condition, the wave vector kx of an incident wave is expressed kx = k0 np sin (2)

Fig. 1. Schematic diagram of variable wavelength SPR sensor.

light goes into the OSA (AQ-4303B, Ando) through the lens and optical ber (3 M Co.). Using the above-mentioned process, the shift in resonant wavelength was measured. Also, we are preparing a new potable wavelength variable SPR system, capable real time measurement and analysis, using small Halogen lamp and spectrum analyzer based on above system. 3.2. Sensor Chip We used a cover glass (n = 1.522, Matsunami Co.) with a similar refractive index to the prism as a substrate. A thin gold layer, which is easy to use for exciting the plasmon on the substrate and chemically safe, was evaporated using a vacuum evaporator. To determine the change of the detecting band on the gold sensor chip manufactured by the above-mentioned method, we evaporated Ta2 O5 using an RF sputtering apparatus. The thickness of the evaporated Ta2 O5 was estimated to be about 300 . When Ta2 O5 was evaporated on the sensor chip, it was possible to choose a resonant wavelength band in the variable wavelength SPR sensor system. Also, by adjusting the thickness of the Ta2 O5 sensing layer, it was possible to measure the sample with high refraction index. The following evaporation conditions of Ta2 O5 were used: an RF power of 100 W, a working pressure of 70 mTorr, a substrate temperature of 100 C and sputtering time of 30 min. The shape of the manufactured sensor chip is shown in Fig. 3. 3.3. Manufacture of Sample and Sensing Layer For immobilizing the salmonella antibody, we prepared 100 ml of salmonella antibody solution at 50 g/ml using 1 mg/ml goat anti-Salmonella solution (KPL). Then we stirred it at room temperature for 12 h while soaking the manufactured sensor chip in this solution. To immobilize penicillinase on the manufactured sensor chip, we immersed the chip in 0.3 M GAmethanol solution for 3 h including 0.4 M 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M N-

and the refraction index of the prism np is expressed as Eq. (3) in relation to the wavelength component np = n( ) = (n ( ) jn ( )) (3)

Also, in the case of the variable wavelength SPR sensor, the resonant phenomenon occurs only in TM-polarized light. Under the same conditions using an incidence variable angle SPR sensor, the wave vector kSPW on the surfaces of the metal and sample is expressed as kSPW k0 metal sample metal + sample (4)

Here, the permittivity , a wavelength-dependent component, is indicated in Eq. (5) in relation to the refraction index = ( ) = ( ) j ( ) = n( )
2

(5)

If kSPW and kx are under the same conditions, the optical energy of the incident wave is converted to that of a surface plasmon wave. Finally, a specic wavelength meeting the condition kSPW = kx produces a resonance, and the intensity of incident light rapidly decrease (Johnston and Mar, 1999). Under this condition, when a specic material is injected into the chamber, the complex permittivity of the thin metal lm is changed by the injected material. Thus, resonance wavelength was shifted by the change in SPW vector. This shift in resonant wavelength is analyzed using the OSA.
3. Experimental 3.1. Conguration of Sensor System The system of this variable wavelength SPR sensor, which detects resonance conditions such as a maximum attenuation wavelength, is shown in Figs. 1 and 2. In this system, we used a white light source (AQ-6315A, Ando) with a wide wavelength band. The light coming out from the light source focused into the prism (BK7, n = 1.515), through a lens and a polarizer (Suruga Seiki Co.), and was polarized in the TM mode. The sensor chip was inserted under the prism. We used matching oil (n = 1.5151.517, Merck) to reduce the difference in the refractive index between the prism (BK7, n = 1.51) and the sensor chip. The sample chamber was manufactured using Teon. The light coming into the prism was totally reected at the thin metal lm of the sensor chip. The reected

Fig. 2. Photograph of fabricated SPR sensor.

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Fig. 3. Schematic diagram of Au/Ta2O5 sensor chip.

Fig. 6. Resonance characteristics of salmonella antibodyantigen reaction.

4. Measurement and Results 4.1. Resonance Characteristics Produced by Salmonella AntibodyAntigen Reaction To evaluate the possible application as a biosensor to the sensor system presented in this paper, we measured SPR phenomena using salmonella, which are food-poisoning bacteria. After immobilizing the salmonella antibody on the sensor chip using concentration of 50 g/ml, we measured the amount of shift in resonant wavelength during the antigenantibody reaction by injecting the salmonella antigen solution at 5 107 cells/ml to the sample chamber for 30 min. Fig. 6 shows the amount of resonant wavelength shift, which is measured for 30 min after injecting the antigen solution to the sample chamber. As in Fig. 7, the resonant wavelength moved linearly for about 10 min with the salmonella antibodyantigen reaction after injecting the antigen solution. We observed weak binding for 1020 min and the absence of binding 20 min after the injection of the salmonella antigen solution. From these results, we found that the reaction between the antibody immobilized on the sensor chip and the antigen injected to the chamber progresses actively for 10 min with the injection. We also found that the shift in resonant wavelength with the salmonella antigenantibody reaction is about 0.26 nm/min.

Fig. 4. Flow chat of penicillinase immobilization.

hydroxysuccinimide (NHS). After cleaning the reacted sensor chip with Phosphate Buffered Saline (PBS) solution, we immobilized penicillinase on the Au/Ta2 O5 sensing layer by putting the device in 0.4 M EDC + 0.1 M NHS + PBS (20) solution for 2 h. Cleaning was performed more than three times with PBS, and we nally immobilized penicillinase on the chip through a reaction in the penicillinase (-lactamase from Bacillus cereus, 10000 units/mg, Aldrich) solution. The ow chart of penicillinase immobilization (Kim et al., 2005; Barie and Rapp, 2001) is shown in Fig. 4, and its schematic diagram is the same as that in Fig. 5.

Fig. 5. Schematic diagrams of penicillinase immobilization.

Fig. 7. Resonance wavelength shift by salmonella antibodyantigen reaction.

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4.2. Resonance Characteristics Produced by Interaction Between Penicillinase and Penicillin After immobilizing penicillinase on the Au/Ta2 O5 sensor chip, we measured the movement of resonant wavelength by injecting 10 mM penicillin solution to the sample chamber. Such measurement was performed similarly to that in the case of salmonella. As a result, we observed that the resonant wavelength increases with time, as shown in Figs. 8 and 9. We also observed a change in the concentration of solution due to the reaction between the penicillinase immobilized on the sensor chip and the injected penicillin. We could make an analogy since there was a rapid reaction between the penicillinase and the penicillin for 15 min and there were no other reactions 15 min after the sample injection. Also, the shift in resonant wavelength due to the penicillinase and penicillin reaction was considered to occur at about 0.8 min1 until the reaction was terminated. Due to the evaporation of Ta2 O5 on the sensor chip, the resonant phenomenon shifted by about 100 nm, compared with that of a non-evaporated Ta2 O5 thin lm: thus, such a phenomenon occurred at 796 nm. On the basis of this phenomenon, it is expected that the sensitivity of sensor systems can be increased and that the samples with high refractive index can be analyzed.

5. Conclusion Most incident light of variable angle SPR sensors using a monochromatic light source have a narrow measurement range and difculties in measuring samples at low concentrations. In this study, we fabricated a variable wavelength SPR sensor and evaluated its characteristics in order to overcome the disadvantages of variable angle SPR sensors. This fabricated sensor measures the shift in resonant wavelength using a white light source and an OSA. From the results of the salmonella antigenantibody and penicillinpenicillinase reaction, we observed a wavelength shift of 0.26 min1 for about 10 min in the salmonella test and the movement of resonant wavelength due to the interaction of penicillin showed a 0.8 min1 shift for 15 min during binding. These results indicate that our variable wavelength SPR sensor is sufciently feasible for measuring a small change in refraction index, detecting low-concentration samples, and measuring biomaterials including clinical diagnosis. For portable and more sensitive wavelength variable SPR system based on these mechanisms, we are making new one using small light source and spectrometer with high resolution using optical ber having the properties of localized SPR. It is thought that wavelength variable SPR will play a greater role in the clinical application needing concentration below 106 M.

Acknowledgments This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (No. KRF2007-357-D00093). This work was supported by Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R11-2008-105-03004-0).

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Fig. 8. Resonance characteristics of penicillin reaction.

Fig. 9. Resonance wavelength shift by penicillinasepenicillin reaction.

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Nyeon-Sik Eum received his Ph.D. in Sensor Engineering from Kyungpook National University, South Korea in 2004. He is currently Postdoctoral at the Bindley bioscuence center, Purdue University, U.S.A. Current research interests include nanoSPR, bio-sensor and micro uidics. Shin-Won Kang received his Ph.D. in Biomedical Science from University of Keio, Japan in 1993. He is currently Professor at the School of Electrical Engineering and Computer Science, Kyungpook National University, South Korea. His research interests are opto-electronic functional device, OLED, bio-sensor and nano device.