3468

DOI 10.1002/pmic.200400879

Proteomics 2004, 4, 34683476

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor
Jong Seol Yuk1, Se-Hui Jung1, Jae-Wan Jung1, Duk-Geun Hong2, Jeong-A Han1, Young-Myeong Kim1 and Kwon-Soo Ha1
1

Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chunchon, Kangwon-Do, South Korea 2 Department of Physics, Kangwon National University, Chunchon, Kangwon-Do, South Korea

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.661025 with the signal fluctuation of 1.061025. The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 mm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.
Keywords: Atomic force microscopy / Protein arrays / Surface plasmon resonance / Wavelength interrogation-based SPR sensor Received: March 14, 2004; revised: April 29, 2004; accepted: May 14, 2004

1 Introduction
Currently, protein arrays have been regarded as powerful tools in proteome researches, since the technology allows the large-scale and high-throughput analysis of proteins in cells, tissues, and organisms. The surface plasmon resonance (SPR) phenomenon has been used as an optical detection technology since Lidberg et al. [1] demonstrated its possibility to use as biosensors in 1983. Biosensors are generally composed of a surface for molecular interactions and a transducer to transfer an optical
Correspondence: Dr. Kwon-Soo Ha, Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chunchon, Kangwon-Do 200-701, South Korea E-mail: ksha@kangwon.ac.kr Fax: 182-33-250-8807 Abbreviations: AFM, atomic force microscopy; EDC, N-ethylN-(dimethylaminopropyl)-carbodiimide; NHS, N-hydroxysuccinimide; SPR, surface plasmon resonance

or electrochemical signal [2, 3]. Typical protein arrays for SPR biosensors are comprised of various proteins immobilized on a chemically modified gold film in the form of dots [4]. The SPR-based sensors are advantageous in the analysis of protein interactions to other sensors based on different technologies, because the SPR spectroscopy allows real-time measurement of molecular interactions without labeling and the optical system for the device is simple [5]. Recently, it has been reported that the SPR phenomenon can be used as a detection method for the analysis of protein arrays [6]. There have been intensive researches on the analysis of biomolecular interactions by SPR sensors based on the angular interrogation [79]. The angular interrogationbased SPR sensors analyze protein interactions by scanning incidence angles at a constant wavelength. There have been also a couple of reports on the analysis of molecular interactions by SPR sensors based on the wavelength interrogation [10, 11]. We have also reported www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 34683476 that the wavelength interrogation-based SPR sensor can be used for the analysis of protein arrays by demonstrating the analysis of protein concentration on protein arrays [12]. However, there are no reports on the analysis of protein interactions on protein arrays by the wavelength interrogation-based SPR sensors, even though the spectral SPR sensors are useful for the high-throughput analysis of molecular interactions. In this study, we present the analysis of multiple protein interactions on protein arrays by using a wavelength interrogation-based SPR sensor. The analysis of protein interactions on the protein arrays was performed by the incubation of C-reactive protein, tissue transglutaminase, hemoglobin, haptoglobin, glutathione S-transferase (GST), GST-RhoA, GST-Rac1, and GST-RhoAN19 with three kinds of antibodies, anti-RhoA, anti-Rac1, and anti-haptoglobin. The Au surface of protein arrays was modifi2ed by the mixed thiol of 11-mercaptoundecanoic acid and mercaptohexanol in order to immobilize proteins. The surface morphology of Au and the thickness of GST-Rac1 immobilized on the mixed thiol film were analyzed by atomic force microscopy (AFM). The wavelength interrogation-based SPR sensor successfully analyzed the shift of resonance wavelength caused by immobilizing various concentrations of GST-Rac1 on a mixed thiol film and the antigen-antibody interactions on the arrays.

Analysis of protein interactions on protein arrays

3469

acterizes the effect of finite thickness of the metal and the presence of prism coupler, and it decreases as the metal thickness increases (Dk 1 ) Dk m ). Dk 1 and Dk m are sp sp sp sp constants when the SPR measurements are performed for the detection of molecular interactions, whereas Dk d sp depends on the dielectric layer of biological molecules [15]. The effect of thickness and dielectric constant of the layer on Dk d is given by sp     2p 2 ed 1ed emr emr 2 1 Dk d d p (2) sp ed 1 1 emr emr l ed where l is the free space wavelength of the light, d is the thickness of dielectric, and ed is the dielectric constant of dielectric on metal. The SPR takes place when the wave vector of a surface plasmon matches with the component of the p-polarized incidence lights wave vector, which is parallel to a metal/dielectric interface (ksp = kx). A surface plasmon excitation, SPR, is observed as a distinctive dip in the reflected light intensity spectrum. The wave vector of the p-polarized incidence light that is parallel to the metal surface is expressed as   2p kx (3) np lsiny l where np is the refractive index of the prism, and y is an incidence angle in the prism.

2.2 Chemicals and reagents

2 Materials and methods

2.1 Theoretical basis of SPR measurement
SPR is an electromagnetic phenomenon in which an evanescent wave excites the charge density oscillation along the metal/dielectric interface. The electromagnetic surface waves, which are exponentially decaying fields normal to the surface, have their maximal intensity on the metal surface. Thus, the change of the optical constant close to the metal surface has the significant influence on the resonance condition, which is the basis of SPR biosensors [13]. When we consider the optical geometry as four layers (prism/metal/dielectric/air), the calculated wave vector of a surface plasmon from a first approximation is given by k sp k 1 Dk m Dk d sp sp sp (1)

Octadecylmercaptan, octadecyltrichlorosilane, mercaptohexanol, 11-mercaptoundecanoic acid, tetracarbone chloride, hexadecane, and ethanolamine were obtained from Sigma (St. Louis, MO, USA). N-Hydroxysuccinimide (NHS), and N-ethyl-N-(dimethylaminopropyl)-carbodiimide (EDC) were from Pierce (Rockford, IL, USA). Monoclonal anti-RhoA and anti-rac1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-haptoglobin, tissue transglutaminase isolated from guinea pig liver, hemoglobin, and haptoglobin were from Sigma. C-Reactive protein was obtained from Scripps Lab (San Diego, CA, USA). GST, GST-RhoA, GSTRhoAN19, and GST-Rac1 were purified by expressing the genes in Escherichia coli (BL21) and purified according to the procedures of Leem et al. [16]. All other chemical reagents were of analytical grade.

2.3 Hydrophobic modification of gold arrays

Gold spot arrays were fabricated and modified according to the procedures of Yi et al. [17]. Briefly, gold arrays with 50 spots (diameter of each spot, 2 mm) were fabricated by depositing Ti/Au (50/450 ) films on Pyrex glasses by www.proteomics-journal.de

where k 1 (2p/l)[em(l)11])1/2 is the wave vector of a sp surface plasmon on the plane surface of a semi-infinite metal with the complex dielectric function em(l) = emr 1 iemi in the absence of a dielectric layer [14]. Dk m charsp

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3470

J. S. Yuk et al.

Proteomics 2004, 4, 34683476 ethanolamine solution (pH 8.6) for 10 min. Then, the protein array was incubated with 10 mg/mL of bovine serum albumin in phosphate buffer for 10 min to reduce nonspecific interactions. The antigen-antibody interactions were investigated by introducing three kinds of antibodies against RhoA, Rac1, and haptoglobin to the eight proteins. The antibody solutions (100 mg/mL) were prepared in a phosphate buffer (8.1 mM Na2HPO4, 1.2 mM KH2PO4, pH 7.4) and every 2 mL of the antibody solutions was applied to each row (with the eight proteins) of protein arrays for 10 min. After incubation, the surfaces were washed twice with 0.1% Tween 20 in phosphate-buffered saline (8.1 mM Na2HPO4, 1.2 mM KH2PO4, 138 mM NaCl, 2.7 mM KCl, pH 7.4) for 5 min, rinsed with dH2O, dried under N2 gas, and immediately analyzed by a wavelength interrogation-based SPR sensor (self-developed).

a RF-magnetron sputtering apparatus at a vacuum of 361026 Torr. The arrays were cleaned by incubation with a cleaning solution of NH4OH:H2O2:H2O (1:1:5 v/v) at 707C for 10 min and washing with dH2O. To prevent protein solutions among spots from being mixed during protein incubation, hydrophobic modification of glass surfaces of Au spots was performed as the previous report [17]. The surfaces of gold spots were coated with a monolayer of octadecylmercaptan by incubation with 5 mM octadecylmercaptan in ethanol for 16 h at room temperature and washing with ethanol. The arrays were then incubated with a mixture of hexadecane/tetracarbone chloride/octadecyltrichlorosilane (20:5:0.04 v/v/v) to generate hydrophobic glass surfaces. After incubation for 20 min, the arrays were washed with a mixture of hexadecane/tetracarbone chloride (20:5 v/v) at 457C, tetracarbone chloride at 457C and ethanol at room temperature in order. Then, octadecylmercaptan layer on the gold spots was removed by incubation with the cleaning solution at 707C for 10 min to make hydrophilic gold surfaces, surrounded by hydrophobic glass surfaces.

2.6 Analysis of protein arrays by the line-scanning mode of a wavelength interrogation-based SPR sensor
The analysis of protein arrays was performed by the linescanning mode of a self-developed wavelength interrogation-based SPR sensor, configured by the Kretschmann geometry of the attenuated total reflection method [14]. A schematic diagram of the SPR sensor is shown in Fig. 1. A 20 W quartz tungsten halogen lamp was used as the light source due to its stable output (Oriel, Stratford, CT, USA). A polarizer was positioned at the input light path to obtain transverse magnetic polarized light. The incidence angle was adjusted by a beam steering device to obtain the resonance wavelength and the incidence angle was set at 487. Protein arrays were coupled with a fused silica prism via an index matching fluid and mounted on an x-y linear stage. Then, the protein arrays were automatically scanned by the line-scanning mode. Reflected light from the protein arrays was collected into an optical fiber whose diameter was 200 mm and analyzed by an AVS-S2000 spectrometer, which provides the spectral resolution of 0.4 nm in the range of 500700 nm (Avantes, Eerbeek, The Netherlands). In order to collimate nonsymmetrical beams, a plano-cylindrical glass lens was positioned in front of the optical fiber. Motion control, data acquisition and analysis, and display were performed by self-developed programs based on LabVIEW software.

2.4 Surface modification of gold arrays with a mixed thiol

Au surfaces were modified with a mixed thiol solution of 1 mM 11-mercaptoundecanoic acid and 2 mM mercaptohexanol in ethanol for 16 h. For amine coupling between the carboxy group of 11-mercaptoundecanoic acid and the amine group of proteins, the carboxy group of 11-mercaptoundecanoic acid was activated by treating the thiol-modified gold surfaces with a solution of 50 mM NHS and 200 mM EDC for 10 min.

2.5 Preparation of protein arrays

For the preparation of protein arrays with various concentrations of GST-Rac1, the protein was serially diluted with 10 mM acetate buffer (pH 4.5) and every 2 mL of the GST-Rac1 solutions was applied by a micropipette to the activated mixed thiol surface of gold array spots for 10 min. Then, the arrays were washed with acetate buffer, rinsed with dH2O, flushed with N2 gas to remove the water, and immediately analyzed by a wavelength interrogation-based SPR sensor [12]. For the analysis of antigen-antibody interactions, 100 mg/mL of eight proteins in 10 mM acetate buffer (pH 4.5), such as C-reactive protein, tissue transglutaminase, hemoglobin, haptoglobin, GST, GST-RhoA, GST-Rac1, and GST-RhoAN19, were applied to the activated mixed thiol surface of gold array spots for 10 min. The residual carboxyl groups of 11-mercaptoundecanoic acids were blocked by incubation with 1 M

2.7 Analysis of surface topology by atomic force microscopy

Surface roughness of a gold film and the thickness of protein layer on the gold arrays were measured by atomic force microscopy (AFM) according to the previous report www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 34683476

Analysis of protein interactions on protein arrays

3471

Figure 1. Schematic diagram of a wavelength interrogation-based SPR sensor. (A) A schematic diagram of a wavelength interrogation-based SPR sensor. Collimated white light enters a polarizer and passes through a right-angle prism contacted with a protein chip at the fixed incidence angle. The reflected light is collected into an optical fiber and analyzed by a spectrometer. (B) Surface structure of protein arrays.

[18]. AFM imaging was performed under air by the contact mode of a Nanoscope IIIa (Digital Instruments, Woodbury, NY, USA). Nanoprobe cantilevers (spring constants of 0.32 N/m) with oxide-sharpened Si3N4 integral tips were used for the contact mode. The applied force was varied from several nN to tens of nN for the contact mode. Film thickness of GST-Rac1 with the mixed thiol film was estimated by measuring the depth of an artificial hole that was made by scratching the layer surface by the contact mode.

3 Results
3.1 Characterization of the wavelength interrogation-based SPR sensor
In order to investigate the applicability of wavelength interrogation-based SPR sensors to the analysis of protein interactions on protein arrays, we constructed a spectral SPR sensor based on the wavelength interrogation (Fig. 1) and studied the shift of resonance wavelength (denoted as SPR wavelength) in response to the protein interactions. Initially, the resonance wavelength on the surface of gold arrays was measured from a SPR spectrum to determine the detection limit of the self-constructed SPR sensor. Detection limit was defined as the minimal difference in refractive index that can be separated by the spectrometer (AVS-S2000) with the resolution of 0.4 nm. As shown in Fig. 2A, the SPR wavelength

Figure 2. (A) SPR spectrum of a gold array and (B) fluctuation of SPR wavelength. (A) SPR reflectivity spectrum on a gold array of Ti/Au (50/450 ). (B) The fluctuation of SPR wavelength was determined from the 100 times measurement of SPR wavelength. www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3472

J. S. Yuk et al.

Proteomics 2004, 4, 34683476 20 to 140 mg/mL. Rac1 is a member of Rho family GTPases and involved in many biological processes including cell growth, vesicle trafficking, and cytoskeletal reorganization [23]. As shown in Fig. 3, seven different SPR spectra were obtained from the centers of array spots coated with different concentration of GST-Rac1 and resonance wavelengths were calculated from the serial SPR spectra. The SPR wavelength was 597.0 nm at 20 mg/mL GST-Rac1 and monotonously elevated by increasing the protein concentration up to 140 mg/mL.

was 588.7 nm, which was determined by the 4th-order polynomial curve fitting technique from an average SPR spectrum of three time measurements. The detection limit of the SPR sensor was expressed by the minimal refractive index variation, which was calculated according to the previous report [19]. The minimal refractive index variation of the SPR sensor was calculated to be 6.661025. The signal variation of the wavelength interrogationbased SPR sensor was determined by monitoring the fluctuation of the SPR wavelength during 100 measurements. The temperature during the measurement constantly maintained at 25.5 6 0.17C, because the SPR wavelength can be affected by the temperature-dependent alteration of bulk medium refractive index [20]. As shown in Fig. 2B, the signal fluctuation was 0.15 nm, corresponding to 1.061025 of refractive index. Thus, the SPR sensor based on the wavelength interrogation is sensitive enough to analyze protein interactions on protein arrays.

3.2 Modification of gold arrays with a mixed thiol

In order to immobilize proteins on gold arrays, the arrays were modified with the mixed thiol of 11-mercaptoundecanoic acid and mercaptohexanol by self-assembled monolayer method. Deposition with the mixed thiol caused the shift of SPR wavelength from 588.7 to 592.8 nm (data not shown), indicating the formation of linker layer on the gold arrays. The changes of resonance signals by the modification with a thin dielectric film are well explained by the changes of refractive index in the metal surface [21, 22]. Next, we have investigated the variation of SPR wavelength between array spots after modifying the gold surface with the mixed thiol, since inter-spot variation is important in the analysis of protein arrays. The variation of SPR wavelength from 50 spots of a protein array was less than 0.7 nm (data not shown), which is close to the resolution (0.4 nm) of AVS-S2000 spectrometer. The small variation of resonance wavelength was possibly contributed by the variation of spot-to-spot metal thickness, which could be occurred during the deposition process [12]. Each resonance wavelength of array spots was used as a reference to monitor protein interactions with the mixed thiol surface.

Figure 3. Changes of SPR spectra with respect to GSTRac1 concentration. (A) SPR spectra were obtained from array spots with the indicated concentrations of GSTRac1 as explained in Section 2. (B) Spectra in (A) were enlarged.

3.3 Changes of SPR wavelength by immobilization of GST-Rac1

In order to characterize the mixed thiol surface for protein immobilization, we have investigated the shift of SPR wavelength as a function of GST-Rac1 concentration from

Then, we have analyzed protein arrays immobilized with various concentrations of GST-Rac1 by the line-scanning mode of the SPR sensor to investigate the distribution of GST-Rac1 on the spots of protein arrays. Each spot was scanned every 100 mm along the central line by moving the x-y stage, which is systemically controlled. This measurement provided detailed characteristics of molecular interactions on the protein arrays. The net shift of SPR wavelengths obtained from the line-scanning analysis, which represented relative amounts of GSTRac1 proteins bound to the mixed thiol surface of the protein arrays, was shown by color spectrum from blue to red. Figure 4A shows the results of the line-scanning analysis of protein arrays, and the increase of GST-Rac1 concentration caused the proportional shift of spectrum color from blue to orange-red. Then, average shifts of SPR wavelength were calculated from Fig. 4A and showed a linear relationship with GST-Rac1 concentration (Fig. 4B). These results suggested that the wavelength interrogation-based SPR sensor could be used as a biosensor for the analysis of protein interactions on protein arrays. www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 34683476

Analysis of protein interactions on protein arrays

3473

3.5 Changes of SPR wavelength and refractive index

It has been reported that the change of SPR response is proportional to the variation of surface concentration of proteins, which is related to the variation of refractive index [27]. We have theoretically calculated the shift of SPR wavelength with respect to the change of the refractive index on the metal surface by using Eqs. (13). In this calculation, we used the effective refractive index of GST-Rac1 (nd = ed1/2), which was defined as the refractive index of the GST-Rac1 layer with the mixed thiol film. We assumed that the refractive index was dependent on the protein concentration and used the effective thickness of GST-Rac1 obtained by the AFM measurement in Fig. 5C. The dielectric constant of Au metal (em), was calculated by fitting the reported values [28]. In Table 1, we have listed the average SPR wavelength of each GST-Rac1 concentration and the corresponding effective refractive index. Average SPR wavelength linearly increased with respect to the change of effective refractive index. These results show that protein concentrations can be analyzed as a function of effective refractive index in the analysis of protein arrays by the wavelength interrogation-based SPR sensor. Table 1. Effective refractive indexes calculated from the average SPR wavelengths of the indicated GSTRac1 concentrations GST-Rac1 concentration (mg/mL) 20 40 60 80 100 120 140 Average SPR wavelength (nm) 597.5 598.9 599.9 600.5 601.1 601.3 602.1 Effective refractive index 1.187 1.225 1.254 1.273 1.291 1.298 1.324

Figure 4. Analysis of protein arrays with the indicated concentrations of GST-Rac1 by the line-scanning mode of the wavelength interrogation-based SPR sensor. (A) Protein arrays were prepared by immobilizing the indicated concentrations of GST-Rac1 proteins on the mixed thiol surface and scanned every 100 mm by the line-scanning mode as explained in Section 2. (B) Averages of SPR wavelength shifts obtained from the color spectra in (A). The results are expressed as means 6 SD from three separate experiments.

3.4 Surface imaging of protein arrays by AFM

The formation of GST-Rac1 layer on the gold surface was studied by AFM to compare with the results obtained by the SPR sensor. First, we observed the surface roughness of gold arrays by the contact mode of AFM because the surface roughness of the metal film affects on the coupling of incident light to the surface plasmon and the change of SPR spectrum [2426]. The surface roughness was approximately 0.8 nm and the surface was uniform enough to be used as an active metal of SPR-based sensors (Fig. 5A). To confirm the formation of the GST-Rac1 layer on the protein arrays, we measured the depth of artificial hole that was made by scratching the protein layer by the contact mode. In these measurements, effective thickness, which is the thickness of protein layer and mixed thiol layer, was measured, since it was very difficult to measure two layers separately. As shown in Figs. 5B and C, the effective thickness of GSTRac1 layers were approximately 2.7 and 3.7 nm at 40 and 80 mg/mL, respectively. Thus, protein concentration affects on the thickness of protein layers on the metal surface.

3.6 Analysis of antigen-antibody interactions on protein arrays by the wavelength interrogation-based SPR sensor
We have demonstrated that the wavelength interrogation-based SPR sensor was sensitive enough to analyze protein arrays. Thus, we have investigated whether the SPR sensor was able to monitor protein interactions on the arrays. To study the possibility, eight kinds of proteins, such as C-reactive protein, tissue transglutaminase, hemoglobin, haptoglobin, GST-RhoA, GST-Rac1, GST-RhoAN19, and GST, were immobilized on the array surface and incubated with three antibodies against RhoA, Rac1, and haptoglobin. The protein arrays were analyzed www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3474

J. S. Yuk et al.

Proteomics 2004, 4, 34683476

Figure 5. (A) Surface roughness of a gold array spot and (B), (C) thickness of GST-Rac1 layers. AFM imaging was performed under air by the contact mode of a Nanoscope IIIa as explained in Section 2. (A) Surface topology of an Au surface. (B) Thickness of 40 mg/mL GST-Rac1 layer. (C) Thickness of 80 mg/mL GST-Rac1 layer.

by the line-scanning mode of the SPR sensor (Fig. 6A). SPR wavelengths obtained by the line-scanning analysis were used to calculate the average SPR wavelengths of each array spot (Fig. 6B). As expected from the color spectra in Fig. 6A, monoclonal anti-RhoA mainly interacted with GST-RhoA and its negative dominant mutant, GST-RhoAN19. Monoclonal anti-Rac1 also strongly bound to GST-Rac1 with a small interaction

with RhoA. Polyclonal anti-haptoglobin showed a significant interaction with haptoglobin, but also bound to hemoglobin with a weaker affinity, indicating the significance of antibody specificity in the array analysis. Thus, it was concluded that the wavelength interrogationbased SPR sensor was good enough to analyze protein-protein or antigen-antibody interactions on protein arrays. www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 34683476

Analysis of protein interactions on protein arrays

3475

Figure 6. Analysis of antigen-antibody interactions on protein arrays by the line-scanning mode. (A) Protein arrays prepared by immobilizing C-reactive protein (CRP), tissue transglutaminase (tTGase), hemoglobin (Hb), haptoglobin (Hp), GST-RhoA, GST-Rac1, GST-RhoAN19, and GST on the mixed thiol, and incubated with antibodies against RhoA, Rac1, and haptoglobin. Then, the protein arrays were analyzed by the line-scanning mode of the wavelength interrogation-based SPR sensor as explained in Section 2. (B) Averages of SPR wavelength shifts obtained from the color spectra in (A). The results are expressed as means 6 SD from three separate experiments.

4 Discussion
We have demonstrated that the wavelength interrogationbased SPR sensor successfully analyzed protein interactions on protein arrays by using the line-scanning mode. The SPR sensor was self-constructed based on the wavelength interrogation. The detection limit, the minimal refractive index variation, of the SPR sensor was calculated to be 6.661025 with a signal fluctuation of 0.15 nm. The formation of the GST-Rac1 layer on the Au surface was verified by the SPR wavelength shift and AFM. Color spectra obtained by the line-scanning mode successfully demonstrated the dose-dependent changes of SPR wavelength shift by GST-Rac1 on protein arrays. The line-scanning mode also successfully demonstrated specific antigen-antibody interactions on protein arrays. Thus, the wavelength interrogation-based SPR sensor can be used as a system for the high-throughput analysis of proteins in cells and tissues. In the present researches, proteins were immobilized by a mixed thiol of 11-mercaptoundecanoic acid and mercaptohexanol on gold arrays. Surface modification of

gold arrays with the mixed thiol film induced an even shift of the SPR wavelength on 50 spots of an Au array with a variation of less than 0.7 nm, indicating that the surface modification with the mixed thiol was a good method for protein array preparation. 11-Mercaptoundecanoic acid was activated with EDC and NHS to immobilize proteins and mercaptohexanol was used as spacer molecules to prevent nonspecific protein interactions. When various proteins, such as tissue transglutaminase, hemoglobin, haptoglobin, GST-RhoA, GST-Rac1, and GST, were introduced to the surface, all proteins showed a significant shift of the SPR wavelength, even though hemoglobin showed an about twice higher shift than other proteins, indicating that the proteins have different binding affinities to the mixed thiol surface. The bound proteins showed specific interactions with their antibodies (Fig. 6), suggesting that mixed thiol is a good surface for protein immobilization with maintaining their activities. We have also investigated whether the mixed thiol surface can be used for the preparation of immunoarrays. Antibodies were well bound to the surface, and the interactions between antibodies and antigens were large www.proteomics-journal.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3476

J. S. Yuk et al.

Proteomics 2004, 4, 34683476 throughput analysis of proteins in cells and tissues. In particular, the line-scanning mode is a useful method to analyze protein arrays by the SPR sensor. This work was supported in part by the grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ10-PG6-GP01-0002).

ly enhanced by the use of protein G as an intermediate layer. Protein G enhanced the interaction of hemoglobin, haptoglobin, GTS, and GST-RhoA with their antibodies by about twofold, except the interaction of GST-Rac1 with anti-Rac1. These results suggested that mixed thiol was a good surface for immunoarrays and the oriented immobilization of antibodies was important for antigenantibody interactions. Thus, the mixed thiol is a good surface for the preparation of protein arrays and immunoarrays. Previously, we have reported the analysis of protein arrays by the spot-scanning mode of the wavelength interrogation-based SPR sensor [17]. Protein arrays can be analyzed very quickly by the spot-scanning mode, since only the central areas of protein array spots are analyzed by the scanning mode. In the present reports, protein arrays were analyzed by the line-scanning mode of the wavelength interrogation-based SPR sensor. The spots of protein arrays were scanned every 100 mm along the central line by systemically moving an x-y stage. The line-scanning mode provided the better understanding of protein interactions on protein arrays, because this analysis method provided detailed information of protein interactions on the horizontal line of protein array spots. It is important to analyze the whole area of protein array spots, since proteins are not uniformly bound to the whole area of array spots. Thus, the line-scanning mode is very useful in the analysis of protein arrays. SPR spectroscopy has various advantages in the analysis of protein interactions, such as real-time measurement of in situ protein interactions and analysis without labeling proteins [5]. In this present report, protein interactions were successfully analyzed without labeling proteins by the wavelength interrogation-based SPR sensor. However, it is known that the SPR spectroscopic method is less sensitive than the labeling method with fluorophores [6]. Thus, several methods have been tried to improve the sensitivity of SPR sensors. A successful method was to use additional antibodies after antigen-antibody interactions. When haptoglobin was applied to the monoclonal anti-haptoglobin surface of protein arrays, only a small shift of SPR wavelength was observed. However, additional incubation with polyclonal anti-haptoglobin, which interacts with haptoglobin, produced a 15 times higher SPR signal than incubation with haptoglobin. Further incubation with anti-rabbit immunoglobulin G (IgG), which interacts with polyclonal anti-haptoglobin, induced an about 36 times higher shift of the SPR wavelength. Thus, the signal amplification by the use of multiple antibodies is a useful method to improve the sensitivity of SPR sensors. In conclusion, the wavelength interrogation-based SPR sensor is a useful system for the high-

5 References
[1] Liedberg, B., Nylander, C., Lundstrm, I., Sens. Actuators B 1983, 4, 299304. [2] Garland, P. B., Quart. Rev. Biophys. 1996, 29, 91117. [3] Scheller, F. W., Wollenberger, U., Warsinke, A., Lisdat, F., Curr. Opin. Biotech. 2001, 12, 3540. [4] Lee, K. B., Park, S. J., Mirkin, C. A., Smith, J. C., Mrksich, M., Science 2002, 295, 17021705. [5] Jnsson, U., Malmqvist, M., Adv. Biosensors 1992, 2, 291 336. [6] Zhu, H., Snyder, M., Curr. Opin. Chem. Biol. 2003, 7, 5563. [7] Salamon, Z., Macleod, H. A., Tollin, G., Biochim. Biophys. Acta 1997, 1331, 131152. [8] Aldinger, U., Pfeifer, P., Schwotzer, G., Steinrcke, P., Sens. Actuators B 1998, 51, 298304. [9] Karlsson, R., Sthlberg, R., Anal. Biochem. 1995, 228, 274 280. [10] Ho, H. P., Wu, S. Y., Yang, M., Cheng, A. C., Sens. Actuators B 2001, 80, 8994. [11] Mu, Y., Zhang, H, Zhao, Z., Song, D., Wang, Z, Sun, J., Li, M., Jin, Q., Sensors 2001, 1, 91101. [12] Yuk, J. S., Yi, S. J., Lee, H. G., Lee, H. J., Kim, Y. M., Ha, K. S., Sens. Actuators B 2003, 94, 161164. [13] Liedberg, B., Nylander, C., Lundstrm, I., Biosensors & Bioelectronics 1995, 10, IIX. [14] Kretschmann, E., Z. Phys. 1971, 241, 313324. [15] Garland, P. B., Quart. Rev. Biophysics 1996, 29, 91117. [16] Leem, S.-H., Shin, I., Kweon, S.-M., Kim, S. I., Kim, J.-H., Ha, K.-S., J. Biochem. Mol. Biol. 1997, 30, 337341. [17] Yi, S. J., Yuk, J. S., Jung, S. H., Zhavnerko, G. K., Kim, Y. M., Ha, K. S., Mol. Cells 2003, 15, 333340. [18] Yuk, J. S., Yi, S. J., Han, J. A., Kim, Y. M., Ha, K. S., Jpn. J. Appl. Phys. 2004, in press. [19] Homola, J., Sens. Actuators B 1997, 41, 207211. [20] Ho, H. P., Wu, S. Y., Yang, M., Cheng, A. C., Sens. Actuators B 2001, 80, 8994. [21] Pockrand, I., Surf. Sci. 1978, 72, 577588. [22] Swalen, J. D., Gorden, J. G., Philpott, M. R., Brillante, A., Pockrand, I., Santo, R., Am. J. Phys. 1980, 48, 669672. [23] Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., Molecular Biology of THE CELL, 4th ed., Garland Science, New York 2002, p. 947. [24] Kapitza, H., Optics Commun. 1976, 16, 7375. [25] Orlowski, R., Raether, H., Surface Sci. 1976, 54, 303308. [26] Orlowski, R., Urner, P., Hornauer, D.-L., Surface Sci. 1979, 82, 6978. [27] Stenberg, E., Persson, B., Roos, H., Urbaniczky, C., J. Coll. Interface Sci. 1991, 143, 513526. [28] Schrder, U., Surf. Sci. 1981, 102, 118130.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.proteomics-journal.de