Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

Biosensors and Bioelectronics 19 (2004) 1497–1504

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium Byung-Keun Oh a , Young-Kee Kim b , Kwang Won Park a , Won Hong Lee a , Jeong-Woo Choi a,∗ a b

Department of Chemical and Biomolecular Engineering, Sogang University, 1 Shinsu-dong, Mapo-gu, Seoul 121-742, South Korea Department of Chemical Engineering, Hankyong National University, 67 Sukjong-dong, Ansung, Kyonggi-do 456-749, South Korea Received 17 September 2003; accepted 3 December 2003

Abstract An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 102 to 109 CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips. © 2003 Elsevier B.V. All rights reserved. Keywords: Immunosensor; Surface plasmon resonance; Protein G; Self-assembly monolayer; Salmonella typhimurium

1. Introduction Diseases caused by food-borne pathogens constitute a world-wide increasing public health problem (WHO, 1995). In the United States, it is estimated that about 76 million illnesses per year are caused by food-borne pathogens (Mead et al., 1999). According to the Centers for Disease Control and Prevention (CDC), the number has increased more than five-fold since 1942. In the Netherlands, the yearly incidence of food-borne diseases is also estimated more than 10 per 100 persons (Notermans and Van de Giessen, 1993). During the last decades the incidence of food-borne diseases has increased in many parts of the world. Salmonella typhimurium, one of the pathogens is mostly found in the intestinal tract of animals such as birds, cattle, and rodents, causes diarrhea, fever, and abdominal pain. It is

∗

Corresponding author. Tel.: +82-2-705-8480; fax: +82-2-711-0439. E-mail address: [email protected] (J.-W. Choi).

0956-5663/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2003.12.009

usually self-limiting, lasting from 2 to 5 days. Human infection has been traced to poultry, dried or frozen eggs, dairy products, shellfish from contaminated water, etc (Shimeld and Rodgers, 1999). Detection of bacterial contamination of food, therefore, is very important for public health protection (Pathirana et al., 2000; Wong et al., 2002). However, conventional microbiological culture methods used for the detection of microorganism are cumbersome and time-consuming, requiring 3–4 days for presumptive results and 5–7 days for confirmation. Most immunoassay techniques, such as radio-immunoassay, fluorescence labeled antibody assay and enzyme-linked immunosorbent assays (ELISA), are widely used, but they are expensive and require time-consuming and complex sample pretreatment procedures. Recently, surface plasmon resonance (SPR) immunosensors have been developed for the measurement of antigens binding to antibody immobilized on the SPR sensor surface, which are capable of detecting analytes in complex biological media with high specificity and sensitivity, with a short detection time, and with simplicity (Darren et al., 1998; Oh

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et al., 2003; Sakai et al., 1998; Toyama et al., 1998). However, the enhancement of sensitivity is required to detect biological materials, as the concentrations of analytes in a biological system are extremely low. The sensitivity of a SPR immunosensor for the detection of antigens with a very low concentration can be increased by control of the orientation of antibodies immobilized on the SPR sensor surface. When antibodies are immobilized on a solid surface, their activity is usually less than that of dispersed antibodies. The main reason for activity reduction is due to the random oriented array of the antibody molecules on the solid surface. Therefore, the development of the immobilization method for antibodies is strongly required to make an oriented layer. Several methods including physical and chemical adsorption have been proposed for preparing an oriented antibody molecular layer on a solid matrix surfaces (Choi et al., 2001a; Kanno et al., 2000; Willfried et al., 1993), and self-assembly techniques have attracted particular attention. Since many of the fundamental and central biological recognition and transduction processes required for immunosensing occur on biological surfaces, particularly within cellular membranes (Breen et al., 1999), it is important to fabricate a bio-mimicking artificial membrane with the best mechanical resistance and functionality of biological molecules. For the construction of a well-defined antibody surface, protein G, a cell wall protein found in most species of Streptococci, can be used for proper orientation of antibody. Since protein G exhibits a specific interaction with the Fc portion of immunoglobulin G (IgG) (Boyle and Reis, 1987), the paratope of IgG can face the opposite side of the protein G-immobilized solid support. As a result, protein G mediated antibody immobilization can lead to a highly efficient immunoreaction. Though the SPR immunosensors for the detection of S. typhimurium have been reported (Koubovà et al., 2001; Bokken et al., 2003), however, the SPR immunosensor using protein G to control the orientation of immobilized antibody molecules has not been reported. Accordingly, the objective of this study is to develop the SPR immunosensor for the detection of S. typhimurium with high sensitivity. In order to endow the orientation of antibody molecules on SPR sensor surface, protein G was introduced. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effects of detergent (Tween-20) for the improvement of binding efficiency of protein G and antibody molecule, and of antibody and antigen due to providing antigen access to the binding site of antibody by separation of antibody molecules clustered around preferred points on the surface or around other antibody molecules were investigated by SPR. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody (Mab) immobilized on self-assembled protein G layer

were performed by atomic force microscope (AFM). Using above methods, the SPR immunosensor for the detection of S. typhimurium using self-assembled protein G layer was developed.

2. Materials and methods 2.1. Materials Protein G (M.W. 22600 daltons) was purchased from Prozyme Inc. (USA). S. typhimurium (KCCM 11806) was kindly donated from the Korean Culture Center of Microorganisms (Korea). E. coli O157:H7 (ATCC 43895) and Yersinia spp. (ATCC 700823) was kindly donated from the American Type Culture Collection (USA). Shigella spp. (KCTC 2517) and Vibrio spp. (KCTC 2715) was kindly donated from the Korean Collection for Type Cultures (Korea). Mab against S. typhimurium was obtained from Biogenesis Ltd. (USA). Other chemicals used in this study were obtained commercially as the reagent grade. 2.2. Immobilization of Mab against S. typhimurium A BK 7 type cover glass plate (18 mm × 18 mm, Superior, Germany) was used as the solid support. The metal coating and substrate cleaning, and the immobilization of biomolecules were performed in the similar way as in the cited reference (Oh et al., 2003). The self-assembled monolayer of 11-MUA on the Au surface was fabricated by submerging the prepared Au substrate into a glycerol/ethanol (1:1, v/v) solution containing 150 mM of 11-MUA for at least 12 h (Yam et al., 2001). For chemical binding between the 11-MUA adsorbed on the Au substrate and the free amine from the protein G, the carboxyl group in 11-MUA was activated by submerging the Au substrate modified with 11-MUA into a solution of 10% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in water/ethanol (10:1, v/v) for 2 h at room temperature. The self-assembled protein G layer was fabricated by the incubation of the activated Au substrate in a solution of protein G in 10 mM phosphate buffer (PBS, pH 7.4) containing 0.14 mol/l NaCl and 0.02% (w/v) thimerosal (PBS) at room temperature for 2 h. Before the immobilization of the antibody, the protein G layer by self-assembly technique on the Au substrate was blocked by inactivating the residual carboxyl group of 11-MUA with 1 M of ethanolamine. To immobilize the Mab, the protein G layer by self-assembly technique was immersed in a solution containing antibodies (100 pmol/ml Mab against BSA and 50 pmol/ml Mab against S. typhimurium) in a PBS buffer. After 4 h of incubation at 4 ◦ C, the surface was rinsed with a PBS buffer. In order to provide antigen access to the binding site of antibody by separation of antibody molecules clustered around preferred points on the surface or around other antibody molecules, Tween-20 was used.

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2.3. SPR spectroscopy 43.6 43.5

SPR angle (deg.)

The bimolecular interactions were monitored using a SPR spectroscope (MultiskopTM , Optrel GbR, Germany) (Harke et al., 1997). The instrumental configuration of the laser light source, polarizer, photo multiplier tube (PMT), and attenuated total reflection (ATR) coupler (Kretschmann, 1971) were the same as in the cited reference (Oh et al., 2002). The resolution of the angle reading of the goniometer was 0.001.

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S. typhimurium was cultivated in a 250 ml flask with 100 ml of medium (medium composition: pancreatic digest of casein 10 g, NaCl 5 g in 1 L deionized water, pH 7.4 ± 0.2 at 25 ◦ C) at 37 ◦ C with shaking at 200 rpm.

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Fig. 1. The effect of increasing concentration on protein G binding to 11-MUA modified Au surface.

2.5. Enzyme-linked immunosorbent assay Wells of a microtiter plate were coated with cell (100 ␮l/well) from S. typhimurium (OD600 nm = 1) and then dried in a 40 ◦ C oven overnight. The wells were washed three times with PBS buffer containing 0.05% Tween-20 (PBS-T; pH 7.4), treated with 300 ␮l/well 5% bovine serum albumin (BSA)–PBS at 37 ◦ C for 1 h for blocking, and then washed with PBS-T. Serial dilutions of Mab to S. typhimurium in PBS-T (1:1,000 to 1:10,000) were prepared and Mab dilutions to wells (50 ␮l/well) were added. After incubation at room temperature for 1 h, they were then washed three times with PBS-T. Diluted secondary antibody horseradish peroxidase (HRP) conjugate was added to each well, and incubated at room temperature for 1 h and then washed five times with PBS-T. Two hundred microliters of substrate solution (0.4 mg/ml of tetramethylbenzidine) was added. The reaction was stopped by the addition of 2N sulfuric acid, and the optical density at 450 nm was measured in a microplate reader.

3. Results and discussion 3.1. Preparation of self-assembled protein G layer The changes of SPR curves by adsorbing 11-MUA, and by binding of protein G in series on Au substrate are shown in Fig. 1. As a result, the SPR angle was shifted significantly from 43.002◦ ± 0.02 to 43.257◦ ± 0.03 by the adsorption of 150 mM 11-MUA on Au surface. And, the SPR angle was shifted from 43.257◦ ± 0.03 to 43.367◦ ± 0.03 by chemical binding between protein G (62.5 nM) and the activated carboxyl group of 11-MUA with EDAC. As the concentration of protein G increased, the shift value of SPR angle was larger and the protein G surface loading was saturated at the concentration of 500 nM. In principle, a surface plasmon resonance is extremely sensitive to the interfacial architecture. An adsorption process leads to a shift in the plasmon

resonance and allows monitoring the mass coverage at the surface with a high accuracy (Fagerstam et al., 1992; Lundstrom, 1994; Matsubara et al., 1988; Salmon et al., 1997). Therefore, the shift in the SPR angle verified that thin layer of 11-MUA on Au surface was formed and protein G molecules were well bound with 11-MUA adsorbed on Au substrate. From above result, the concentration of protein G for antibody immobilization was determined to 500 nM. AFM images of the 11-MUA layer on the Au substrate and the self-assembled protein G layer on the 11-MUA layer in comparison with that of bare gold are shown in Fig. 2. Since 11-MUA has a long alkyl chain, which can provide van der Waals attractive force among the molecules, it can form a strong and close-packed 2D molecular array (Nelles et al., 1998; Choi et al., 2001b). A fairly well organized molecular array could be observed in nm scale. Also, it could be observed that protein G molecules are adsorbed onto the Au substrate modified with 11-MUA as an aggregated pattern in solid-like state with keeping its random cloud-like structure as in bulk solution. From the above results, it can be concluded that the self-assembled protein G layer was fabricated on the Au substrate. Many investigators have washed their preparation with detergents (usually Tween-20) after protein immobilization for providing analytes access to the binding site of immobilized protein. Fig. 3a and b show the AFM image variations of self-assembled protein G layer by Tween-20 treatment. And, Fig. 3c represents the shift degree of SPR minimum by self-assembled protein G–antibody (Mab against BSA) complex formation with/without Tween-20 treatment of self-assembled protein G layer on 11-MUA modified Au surface. As shown in Fig. 3a and b, the cluster size of self-assembled protein G bound to 11-MUA modified Au surface is decreased by Tween-20 washing. And, as might have been expected, the shift of SPR angle by antibody

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Fig. 2. AFM images of bare gold, 11-MUA layer, and self-assembled protein G layer: (a) bare Au (scan size: 0.3 ␮m × 0.3 ␮m), (b) 11-MUA (scan size: 5 Å × 5 Å), (c) self-assembled protein G layer (scan size: 0.3 ␮m × 0.3 ␮m).

binding to self-assembled protein G layer before Tween-20 washing was smaller than that after Tween-20 washing. The variation of SPR angle by the binding interaction between protein G and antibody (100 nM Mab against BSA) is 0.225◦ and 0.315◦ , respectively. This results can be caused by the function of detergent as follows: (a) complete dissociation of molecules that are bound with a low free energy; (b) separation of protein G molecules clustered around preferred points on the surface or around other protein G molecules. The above results show that the binding site of the protein G is more accessible to antibody binding by Tween-20 treatment. 3.2. Antibody immobilization on self-assembled protein G layer The change of SPR curve by adsorbing antibody (100 nM of Mab against BSA) on self-assembled protein G layer is shown in Fig. 4. The SPR angle was shifted significantly from 43.437◦ ± 0.03 to 43.752◦ ± 0.03 by the immobilization of antibody on self-assembled protein G layer, because a shift of SPR angle resulted from the adsorption of dielectric materials on SPR sensor surface. From this result, the binding interaction between self-assembled protein G layer and antibody molecules could be confirmed. Fig. 5a and b show the AFM image variation of antibody layer by Tween-20 treatment. And, Fig. 5c represents

the shift degree of SPR angle by antibody (100 nM Mab against BSA)—its antigen (100 nM BSA) complex formation with/without Tween-20 treatment of antibody layer immobilized on self-assembled protein G layer. As shown in Fig. 5, the aggregated pattern size of the antibody molecules bound to self-assembled protein G layer was decreased by Tween-20 washing. And, as might have been expected, the shift of SPR angle by antigen (100 nM BSA) binding to antibody (100 nM Mab against BSA) layer with Tween-20 treatment was larger than that without Tween-20 treatment. The variation of SPR angle by the binding interaction between antibody and its antigen was 0.36◦ and 0.18◦ , respectively. The above results show that the binding sites of the antibody are more accessible to antigen binding by Tween-20 treatment. The effect of protein G about the binding interaction between antibody and antigen was investigated in comparison with shift degree of SPR angle by binding of antigen to immobilized antibody on Au substrate without/with protein G. The results were shown in Fig. 6. The variation of SPR angle by the binding interaction between antibody and antigen without/with protein G is 0.195◦ and 0.36◦ , respectively. Compared with the shift degree of SPR angle by binding interaction between antibody and antigen, the shift degree of SPR angle in case of immobilized antibody on solid surface using protein G is larger than that of SPR angle in case of directly immobilized antibody on solid surface without

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Fig. 3. The effect of detergent (Tween-20) in self-assembled protein G–antibody complex formation: (a) AFM image of protein G layer before Tween-20 treatment (scan size: 0.1 ␮m × 0.1 ␮m), (b) AFM image of protein G layer after Tween-20 treatment (scan size: 0.1 ␮m × 0.1 ␮m), and (c) SPR angle shift by antibody binding to self-assmebled protein G layer (a: protein G layer, b: antibody layer).

protein G. The result of Fig. 6 suggests that the amount of antibody–antigen complex formation is higher by using protein G, since the binding site of immobilized antibody on solid surface is exposed to the medium of the analytical system.

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3.3. Surface plasmon resonance immunosensing for detection of S. typhimurium

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Fig. 4. The change of the SPR curve by antibody binding to self-assembled protein G layer (a: protein G layer, b: antibody layer).

The selection of antibody with high specificity is important in developing the immunosensor, because the specificity for the measurement of analytes in all immunosensor system is dependent on used antibody. In this study, commercial Mab against S. typhimurium was used. In order to investigate the cross-reaction between commercial Mab against S. typhimurium and related pathogens existed in contaminated water, indirect ELISA was performed as shown in Fig. 7. As a result, it was observed that Mab against S. typhimurium had the high specificity with corresponding pathogen and

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Fig. 5. The effect of detergent (Tween-20) in antibody–antigen complex formation: (a) AFM image of antibody layer before Tween-20 treatment (scan size: 0.3 ␮m × 0.3 ␮m), (b) AFM image of antibody layer after Tween-20 treatment (scan size: 0.3 ␮m × 0.3 ␮m), and (c) SPR angle shift by antigen binding to immobilized antibody layer (a: antibody layer, b: antigen layer). 1.5 Salmonella spp. E. coli O157:H7 Yersinia spp. Shigella spp. Vibrio spp.

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Fig. 6. The effect of protein G in antibody–antigen complex formation.

Fig. 7. The result of indirect ELISA for Mab against S. typhimurium with various pathogens.

SPR angle shift (deg.)

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fabrication technique of a SPR immunosensor for the detection of S. typhimurium could also be applied to construct other immnosensors or protein chips with a high efficiency.

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Acknowledgements This study was supported by a grant from the International Mobile Telecommunications 2000 R&D Project, Ministry of Information & Communication, Republic of Korea (01-PJ11-PG9-01NT00-0034).

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References

The concentration of S. typhimurium (CFU/mL)

Fig. 8. The changes of the SPR angle shift by binding between immobilized Mab against S. typhimurium and various concentrations of S. typhimurium.

did not react with various related pathogens such as E. coli O157:H7, Yersinia spp., Shigella spp., and Vibrio spp. As such, it was deemed to be appropriate as an antibody for the detection of S. typhimurium. The signal relationship with respect to the pathogen concentration is presented in Fig. 8. As shown in Fig. 8, the shift in the SPR angle was also increased in proportion to the concentration of S. typhimurium, thereby presenting a linear relationship between the concentration of S. typhimurium and the SPR angle shift. The lowest detection limit for the immunosensor based on SPR was 102 CFU/ml, plus the assay was four orders of magnitude more sensitive than a standard ELISA (Kim et al., 1999). Accordingly, it was concluded that an immunosensor based on SPR can be used to monitor S. typhimurium. 4. Conclusions An immunosensor based on SPR was developed for the detection of S. typhimurium with high sensitivity by controlling the orientation of antibody molecules immobilized on SPR surface using self-assembled protein G. A self-assembled protein G layer on Au surface was fabricated by adsorbing 11-MUA and an activation process for chemical binding between free amine (–NH2 ) of protein G and 11-MUA using EDAC in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The binding efficiency of protein G and antibody molecule, and of antibody and antigen were improved by using detergent such as Tween-20. The surface morphology analyses of self-assembled protein G layer on Au substrate and Mab immobilized on self-assembled protein G layer were performed by AFM. Consequently, an immunosensor based on SPR for the detection of S. typhimurium using self-assembled protein G was developed with a detection range of 102 to 109 CFU/ml. The current

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