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Surface modification of thin polyion complex film for surface plasmon resonance immunosensor
Available online at www.sciencedirect.com
Sensors and Actuators B 130 (2008) 320–325
Surface modification of thin polyion complex film for surface plasmon resonance immunosensor Ryoji Kurita a,∗ , Yoshiki Hirata a , Soichi Yabuki a , Yoshimi Yokota a , Dai Kato a , Yukari Sato a , Fumio Mizutani b , Osamu Niwa a a
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, Japan b University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan Available online 10 August 2007
Abstract A surface plasmon resonance-based immunosensor was developed by modifying the surface of a thin polyion complex film consisting of polyl-lysine and poly-styrenesulfonate. The suppression of non-specific protein adsorption was investigated by changing the blend ratio of the two polymers. The protein adsorption fell to less than 10% that of an unmodified surface when the poly-l-lysine to ratio was 4:6. The binding constant between immobilized B-type natriuretic peptide on the polyion complex film and its antibody was calculated to be 1.1 × 108 (M−1 ), which is a satisfactory level for immuno-affinity on a solid surface. We also obtained stable and reproducible responses without losing the affinity on the polyion complex film. © 2007 Elsevier B.V. All rights reserved. Keywords: Surface plasmon resonance; Immunosensor; Polyion complex film; Protein adsorption
1. Introduction Surface plasmon resonance (SPR) measurement is a surface sensitive analytical technique that has been successfully incorporated into an immunosensor format for the non-labeled assay of various biomolecules [1–5]. The SPR immunosensor also has advantages as regards bedside monitoring and point-of-care testing (POCT) because the method is a rapid, simple, safe and low power technique. However, its major disadvantage is its poor sensitivity to small molecules and poor selectivity resulting from non-specific adsorption. In principle, the sensitivity of the SPR immunosensor depends on the molecular weight of the analytes. Thus, the sensitivity to small molecules is lower than that of other optical methods such as conventional fluorescence detection because small molecule bindings have little effect on the refractive index on a sensing surface. For example, the detection limit of the SPR immunoassay is reported to be around several tens of nM for small peptides [6,7]. In addition, some immobilized antibod-
∗
Corresponding author. Tel.: +81 29 861 6166; fax: +81 29 861 6177. E-mail address: [email protected] (R. Kurita).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.08.007
ies are unable to form stable layers because they are denatured to lose their specific activity compared with that of free antibodies [8]. Very recently, we developed a highly sensitive enzymelabeled SPR immunosensor using an antibody labeled with acetylcholine–esterase, which produces thiol molecules [9]. The detection limit was greatly improved by the thiol accumulation effect in a micro-flow channel, and this proposed immunosensor is one of the most sensitive sensors yet reported based on an SPR detection system. On the other hand, non-specific adsorption on the sensing surface, especially protein fouling [10,11], is a serious problem when measuring trace level biomolecules in real samples using a conventional SPR immunosensor, which detects an SPR angle shift caused by the formation of an immuno-complex on the sensing surface. This is because the SPR angle is increased by the non-specific protein adsorption, which makes it difficult to detect small SPR angle shifts caused by an immuno-reaction with a small amount of analyte. Various kinds of films and modification methods have been studied to suppress the non-specific adsorption that occurs during SPR measurements [12–14], for example, dextran, polyethylene glycol [15], and poly-vinylalcohol [16]. For a highly sensitive and selective SPR immunosensor, it would be better
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2. Experimental
vated the film for 15 min. We then added 1 L of 2 mg/mL BNP (in acetate buffer) to the buffer on the polyion complex film and stirred it for a few seconds with a micropipette, followed by 15 min incubation. After rinsing with acetate buffer, we quenched any unreacted NHS-activated groups by incubation for 5 min with 0.1 M aminoethanol (in acetate buffer). All immobilization reactions were performed at room temperature. The PDMS cover was formed with previously reported procedures [20]. The master pattern was fabricated on a glass wafer by a conventional photolithographic technique with a positive photoresist (PMER P-LA900PM, Tokyo Ohka Kogyo, Japan). Liquid PDMS oligomer (Dow Corning Asia, Japan) and a crosslinking agent were mixed and poured onto the master with the flow channel pattern. After curing the mixture for 60 min at 60 ◦ C, the PDMS layer was removed from the master.
2.1. Reagents
2.3. Measurements using immunosensor
Poly-l-lysine hydrobromide (average M.W. 80,000) was purchased from Sigma (St. Louis, MO). Poly-(sodium 4styrenesulfonate) (average M.W. 70,000) and bovine serum albumin were purchased from Aldrich. 0.1 M phosphate buffer (pH 7.0) and glycine were purchased from Nakalai (Kyoto, Japan). N-hydroxysuccinimide (NHS) and N-ethyl-N (3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) were purchased from Pierce (Rockford, IL). Human B-type natriuretic peptide-32 (BNP) was purchased from Phoenix Pharmaceuticals (Melmont, CA). Mouse monoclonal anti-human B-type natriuretic peptide-32 antibody (anti-BNP antibody) was purchased from ABCAM (Cambridge, UK). Polymer solutions were prepared with Millipore water (Millipore Co.).
Before undertaking any measurements, we peeled the adhesive sheet from the glass plate, and attached the plate to the PDMS cover with the micro-channel. We then connected inlet and outlet tubes (0.12 mm i.d., 0.7 mm o.d., BAS, Japan) to the PDMS cover. We connected the other end of the outlet tube to a syringe, which was equipped with a syringe pump (CMA, Sweden). The sample solution was injected via the sample tube in the suction mode at a flow rate of 2 L/min. First, we introduced a phosphate buffer solution (blank solution) to obtain a stable baseline, then we introduced the sample solution and monitored the SPR angle shift value (θ SPR ). We obtained an SPR curve and measured its minimum reflection angle (SPR angle) using portable SPR equipment (NTT-AT, Japan), which was developed in collaboration with one of the present authors. Index matching oil (n = 1.510, Cargille Laboratories Inc.) was used to obtain optical contact between the glass plate of the immunosensor and the prism on the SPR equipment.
if the membrane on the sensing surface had both a less nonspecific adsorption property and a high density of functional groups for immobilizing antibodies (or antigens) without losing their specific binding activity. In this study, we report the surface modification of an SPR immunosensor with a thin polyion complex film consisting of poly-l-lysine and poly-styrenesulfonate. We optimized the modification of the film and studied the effects of the non-specific adsorption of protein, especially albumin, which is found in large amounts in blood, using SPR and AFM while varying the thickness and blend ratio for the film formation. We also measured the binding constant between antigens and antibodies on the polyion complex film.
2.2. Fabrication of immunosensor with polyion complex film The immunosensor consists of a glass plate (16 mm × 16 mm) with a thin gold film, and a poly-dimethylsiloxane (PDMS) cover with a micro-channel (20 m deep, 2 mm wide). Thin titanium was deposited on the glass plate by using an RF sputtering machine, and gold film was then formed without breaking the vacuum [17]. The total thickness of the gold and titanium film was 50 ± 2 nm. We then sealed the glass plate using a polymeric adhesive sheet with a 3 mm diameter hole. Next, we placed a mixture solution of poly-l-lysine and polystyrenesulfonate on the gold film on the plate, and allowed the plate to dry for one night at room temperature [18]. The polyl-lysine and poly-styrenesulfonate concentrations were varied from 0.62 pg/mm2 to 62 ng/mm2 to obtain films with different thicknesses. We also changed the blend ratio of the polyion complex film from 7:3 to 1:9 by changing the amounts of both solutions. The total volume of solution loaded on each plate was 10 L (1.4 L/mm2 ). When we examined the immuno-affinity on the polyion complex film, we used the following common carbodiimide coupling reaction provided by an EDC/NHS system [19]. We placed 9 L of 10 mM acetate buffer (pH 5.5) containing 0.4 mg/mL EDC and 1.1 mg/mL NHS on the polyion complex film, and acti-
3. Results and discussions 3.1. Polyion complex film formation for SPR measurement Fig. 1(a) and (b) shows the respective variations in the SPR curve shape and the SPR angle for distilled water when we varied the concentrations of the loaded polyion complex solution for film formation. The SPR angles were around 67.5◦ , and narrow SPR curves were observed when the polymer concentration was less than 1 ng/mm2 . However, the SPR angle became large and the SPR curve broadened when the concentrations were greater than 1 ng/mm2 . Fig. 2 shows AFM images of the polyion complex film on the gold surface. The gold surface was covered with the film, and flat hill-like structures were observed in the AFM images. When the polymer concentration was increased, many grains were observed and this caused the SPR angle increase. This indicates that a solution with a high polyion complex concentration is inappropriate as regards forming a film for SPR measurement because a high concentration solution tends to form many grains rather than a thin film on the gold surface. AFM observation revealed that the film
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Fig. 1. (a) SPR curves when the concentrations of the loaded polyion complex solution on a gold surface were 0.62, 6.2, 62 ng/mm2 , respectively. (b) Variation in SPR angle for distilled water when the polyion complex solution was changed over a wide range.
Fig. 2. AFM images (10 m × 10 m) of a gold surface modified with the polyion complex film when the polyion complex solutions were varied. (a) 0.62 ng/mm2 , (b) 2.48 ng/mm2 , (c) 3.72 ng/mm2 . The height profiles are given beneath each AFM image.
thickness was around 5 nm when the polymer concentration was around 1 ng/mm2 . Based on the AFM observation and the SPR angles in Fig. 1, we chose a film formed with a concentration of 0.62 ng/mm2 . This is because a thin polymer layer is advantageous for SPR measurement since the intensity of an evanescent field decreases with distance from the gold surface. The polymer was much thinner than previously reported modified polymers, other than those where the self-assembly technique was used.
adsorption was observed when the film composition was slightly poly-styrenesulfonate rich and weakly anionic charged. This could suggest that electrostatic repulsion contributes slightly to the rejection of albumin, since it is an anionic protein in a neutral solution.
3.2. Non-specific adsorption properties of polyion complex film Fig. 3 shows the variation in the SPR angle shift value (θ SPR ) for 1 mg/mL albumin solution injection when we varied the blend ratio of the polyion complexes used for film formation. The θ SPR was almost the same as that of an unmodified gold surface for very different blend ratios. However, protein fouling was suppressed when the poly-l-lysine to poly-styrenesulfonate blend ratio was between 5:5 and 3:7. Non-specific adsorption was well suppressed when the blend ratio was 4:6, and was less than 10% that of bare gold film. Many researchers have reported various non-protein adsorption polymers, which are neutral and hydrophilic. Our polyion complex film also suppresses protein adsorption when the polyanion and polycation blend content was almost the same. However, the minimum
Fig. 3. Variation in the non-specific adsorption for 1 mg/mL of albumin when the poly-l-lysine to poly-styrenesulfonate blend ratio is varied. Albumin solution was injected for 5 min at a flow rate of 2 L/min.
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Fig. 4. (a) SPR sensorgrams when 1–100 g/mL of anti-BNP antibody solutions was introduced onto the polyion complex film modified with BNP. (b) Scatchard plot analysis of bound anti-BNP antibody against bound/free anti-BNP antibody obtained from Fig. 4(a).
Based on the θ SPR , about 90 pg/mm2 of albumin was adsorbed on the polyion complex film when the blend ratio was 4:6 since a 1◦ SPR angle shift is reported to correspond to a protein adsorption of 10 ng/mm2 [21,22]. The 90 pg/mm2 albumin adsorption is almost equivalent to the amount of non-specific albumin adsorption on a commercially available dextran film, which is one of the most widely used films for SPR measurement [23]. Although the amount of adsorbed albumin reported in this paper is slightly larger than that of synthesized polymers designed to suppress non-specific protein adsorption such as ␣-acetal--mercapto-poly-ethyleneglycol (24 pg/mm2 ) [24] and poly-sulfobetaine methacrylate (38 pg/mm2 ) [25], we are able to obtain the film with a simple and rapid process solely by mixing two common commercially available polymers. Furthermore, it is difficult to modify poly-ethylene glycol with an antigen or antibody because it contains no functional groups, such as amino or carboxyl groups, which are commonly used to immobilize antibodies or antigens. In contrast, our polyion complex film is advantageous for an SPR immunosensor since the film has both amino and carboxyl groups. In this study, we have focused particularly on the albumin rejection among proteins for future POCT application because blood samples contain a large amount of albumin. The polyion complex film may also be effective for rejecting other proteins since the charge in the
polymer film can be tuned by changing the poly-l-lysine to poly-styrenesulfonate blend ratio, however, this requires further investigation. 3.3. Immuno-affinity on polyion complex film An immunosensor with a low detection limit can be obtained by increasing the amount of antibody or antigen without losing its activity in addition to low non-specific adsorption. Therefore, in the next step, we examined the binding constant of immunoaffinity on the polyion complex film because it is known that antibodies or antigens immobilized on a solid surface sometimes lose their specific activity. Fig. 4(a) shows SPR sensorgrams when we injected 1 to 100 g/mL of anti-BNP antibody solution for 10 min into an immunosensor prepared by immobilizing BNP on the polyion complex film. We observed a large θ SPR as the antibody concentration increased. We also obtained a Scatchard plot as shown in Fig. 4(b) from the SPR sensorgrams of 5–100 g/mL antibody solutions in Fig. 4(a) except with 1 and 2 g/mL antibody injections. This exception is because the two SPR sensorgrams did not remain in a steady state during the 10 min antibody injection, i.e. the antigen–antibody reaction on the polyion complex film did not reach equilibrium. The binding constant of the immuno-reaction was estimated to be
Fig. 5. (a) SPR sensorgrams for 10 g/mL anti-BNP antibody solutions containing 0, 0.04, 0.4, 4, 40, 400 g/mL BNP on polyion complex film modified with BNP. (1) Baseline for blank buffer. (2) Injection of a mixture solution of BNP and anti-BNP antibody. (3) Injection of buffer and θ SPR measurement. (4) Regeneration with glycine–HCl. (b) Calibration curve for BNP. Y-axis is θ SPR at 3rd step obtained from Fig. 5(a).
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Fig. 6 shows the SPR sensorgrams we obtained when we injected a mixture solution of 10 g/mL anti-BNP antibody and 400 ng/mL BNP into the immunosensor five times after regeneration with glycine–HCl buffer (pH 2.0). The heights and shapes of the SPR sensorgrams were almost the same and we observed no reduction in the response. The relative standard deviation in the θ SPR for the five measurements was 3.4%. We were able to obtain stable and reproducible responses using our immunosensor with the thin polyion complex film. 4. Conclusion
Fig. 6. SPR sensorgrams when a mixture solution of BNP and anti-BNP antibody was injected repeatedly after regeneration with glycine–HCl buffer. Filled and open bars indicate the injection of the mixture solution and glycine–HCl buffer, respectively.
1.1 × 108 (M−1 ). This value is a satisfactory level for immunoaffinity on a solid surface [26,27]. We believe this to be because polyion complex film has a high biocompatibility without denaturation. Fig. 5(a) shows SPR sensorgrams for 10 g/mL anti-BNP antibody solutions on the BNP immobilized polyion complex film surface after reacting with sample BNP with concentrations ranging from 0 to 400 g/mL. We introduced a blank buffer solution for 1 min onto the BNP modified polyion complex film, and obtained a stable baseline as the first step (1). Then we introduced a mixture solution of BNP and anti-BNP antibody onto the sensing surface for 5 min, after 20 min of incubation in a microcuvetto as the second step (2). In this step, the reacted antibodies are not bound to the BNP on the polyion complex film. In contrast, only unreacted anti-BNP antibodies bound to the BNP on the film and cause an SPR angle increase. In the third step, we again introduced a buffer solution for 3 min and measured the θ SPR (3). Finally, we regenerated the film with an injection of glycine–HCl buffer (pH 2.0) for 3 min (4). Fig. 5(b) shows the calibration curve for BNP obtained from SPR sensorgrams in Fig. 5(a). We observed a large θ SPR when the BNP concentration was low. However, the θ SPR decreased as the BNP concentration increased. This is because the unreacted antibody decreased as the sample BNP concentration increased, so the amount of bounded antibody on the polyion complex film decreased. The detection limit for the BNP concentration with our immunosensor was around a few tens of ng/mL (about 1 nM). This value is about one order of magnitude better than that obtained for previously reported non-labeled SPR immunosensors for small peptides [6,7]. A non-labeled SPR immunosensor usually detects SPR angle shifts caused by small analytes binding with the antibody modified sensing surface. Our immunosensor is advantageous in terms of improving the sensitivity since we measured unreacted antibodies whose molecular weight (150 kDa) was larger that that of BNP (3465). This was achieved by the highly suppressing property of the non-specific adsorption of reacted antibodies on the polyion complex film.
This paper describes the modification of a gold sensing surface for an SPR-based immunosensor with a thin polyion complex film consisting of poly-l-lysine and polystyrenesulfonate. The film suppresses non-specific protein adsorption when the film thickness and the blend ratio of the two polymers are optimized. Biomolecules such as small peptides can also be immobilized on our film with a relatively high density without losing binding activity. The polyion complex film is beneficial for modifying the sensing surface of a non-labeled direct SPR immunosensor. Although we describe only BNP monitoring, the film could be applied to various biomolecules by changing both the antigen on the film and its antibody. References [1] V. Kanda, J.K. Kariuki, D.J. Harrison, M.T. McDermott, Label-free reading of microarray-based immunoassays with surface plasmon resonance imaging, Anal. Chem. 76 (2004) 7257–7262. [2] B. Fitzpatrick, R. O’Kennedy, The development and application of a surface plasmon resonance-based inhibition immunoassay for the determination of warfarin in plasma ultrafiltrate, J. Immunol. Methods 291 (2004) 11–25. [3] T. Akimoto, K. Ikebukuro, I. Karube, A surface plasmon resonance probe with a novel integrated reference sensor surface, Biosens. Bioelectron. 18 (2003) 1447–1453. [4] K.V. Gobi, C. Kataoka, N. Miura, Surface plasmon resonance detection of endocrine disruptors using immunoprobes based on self-assembled monolayers, Sens. Actuators B: Chem. 108 (2005) 784–790. [5] T. Uda, K. Inoue, T. Nishimura, E. Hifumi, K. Shimizu, N. Egashira, Simultaneous detection of two antigens by surface plasmon resonance sensor, Electrochemistry 69 (2001) 976–979. [6] P. Gomes, D. Andreu, Direct kinetic assay of interactions between small peptides and immobilized antibodies using a surface plasmon resonance biosensor, J. Immunol. Methods 259 (2002) 217–230. [7] R.P.H. Kooyman, H. Kolkman, J. Van Gent, J. Greve, Surface plasmon resonance immunosensors: sensitivity considerations, Anal. Chim. Acta 213 (1988) 35–45. [8] B. Oh, W. Lee, Y. Kim, W.H. Lee, J. Choi, Surface plasmon resonance immunosensor using self-assembled protein G for the detection of salmonella paratyphi, J. Biotechnol. 111 (2004) 1–8. [9] R. Kurita, Y. Yokota, Y. Sato, F. Mizutani, O. Niwa, On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system, Anal. Chem. 78 (2006) 5525–5531. [10] R. Kurita, H. Tabei, Y. Iwasaki, K. Hayashi, K. Sunagawa, O. Niwa, Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane, Biosens. Bioelectron. 20 (2004) 518–523. [11] R. Kurita, N. Yabumoto, O. Niwa, Miniaturized one-chip electrochemical sensing device integrated with a dialysis membrane and double thin-layer flow channels for measuring blood samples, Biosens. Bioelectron. 21 (2006) 1649–1653.
R. Kurita et al. / Sensors and Actuators B 130 (2008) 320–325 [12] V. Silin, H. Weetall, D.J. Vanderah, SPR studies of the nonspecific adsorption kinetics of human IgG and BSA on gold surfaces modified by self-assembled monolayers (SAMs), J. Colloid Interface Sci. 185 (1997) 94–103. [13] M. Mrksich, G.B. Sigal, G.M. Whitesides, Surface-plasmon resonance permits in-situ measurement of protein adsorption on self-assembled monolayers of alkanethiolates on gold, Langmuir 11 (1995) 4383–4385. [14] S. Toyama, A. Shoji, Y. Yoshida, S. Yamauchi, Y. Ikariyama, Surface design of SPR-based immunosensor for the effective binding of antigen or antibody in the evanescent field using mixed polymer matrix, Sens. Actuators B: Chem. 52 (1998) 65–71. [15] J.M. Brockman, A.G. Frutos, R.M. Corn, A multistep chemical modification procedure to create DNA arrays on gold surfaces for the study of protein-DNA interactions with surface plasmon resonance imaging, J. Am. Chem. Soc. 121 (1999) 8044–8051. [16] D.A. Barrett, M.S. Hartshorne, M.A. Hussain, P.N. Shaw, M.C. Davis, Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance, Anal. Chem. 73 (2001) 5232–5239. [17] R. Kurita, H. Tabei, Z. Liu, T. Horiuchi, O. Niwa, Fabrication and electroproperties of an interdigitated array electrode in a microfabricated wall-jet cell, Sens. Actuators B: Chem. 71 (2000) 82–89. [18] S. Yabuki, F. Mizutani, Y. Hirata, Preparation of D-amino acid oxidaseimmobilized polyion complex membranes, Sens. Actuators B: Chem. 76 (2001) 142–146. [19] M.R. Lewis, A. Raubitschek, J.E. Shively, A facile water-soluble method for modification of proteins with DOTA: use of elevated-temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled, Bioconjug. Chem. 5 (1994) 565–576. [20] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Rapid prototyping of microfluidic systems in poly(dimethylsiloxane), Anal. Chem. 70 (1998) 4974–4984. [21] L.G. Fagerstam, A. Frostellkarlsson, R. Karlsson, B. Persson, I. Ronnberg, Biospecific interaction analysis using surface-plasmon resonance detection applied to kinetic, binding-site and concentration analysis, J. Chromatogr. 597 (1992) 397–410. [22] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitativedetermination of surface concentration of protein with surface-plasmon resonance using radiolabeled proteins, J. Colloid Interface Sci. 143 (1991) 513–526.
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[23] R.A. Frazier, G. Matthijs, M.C. Davies, C.J. Roberts, E. Schacht, S.J.B. Tendler, Characterization of protein-resistant dextran monolayers, Biomaterials 21 (2000) 957–966. [24] K. Uchida, H. Otsuka, M. Kaneko, K. Kataoka, Y. Nagasaki, A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: the substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption, Anal. Chem. 77 (2005) 1075–1080. [25] Y. Chang, S. Chen, Z. Zhang, S. Jiang, Highly protein-resistant coatings from well-defined diblock copolymers containing sulfobetaines, Langmuir 22 (2006) 2222–2226. [26] M.H.F. Meyer, M. Hartmann, M. Keusgen, SPR-based immunosensor for the CRP detection—a new method to detect a well known protein, Biosens. Bioelectron. 21 (2006) 1987–1990. [27] Y. Kikuchi, S. Uno, M. Nanami, Y. Yoshimura, S. Iida, N. Fukushima, M. Tsuchiya, Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance, J. Biosci. Bioeng. 100 (2005) 311–317.
Biographies Ryoji Kurita received his PhD degree from Kyushu University in 2004. Yoshiki Hirata received his PhD degree from Tokyo Institute of Technology in 1990. Soichi Yabuki received his PhD degree from Tokyo Institute of Technology in 1989. Yoshimi Yokota received her MS degree from Kyoto University in 1988. Dai Kato received his PhD degree from Kumamoto University in 2003. Yukari Sato received her PhD degree from Hokkaido University in 1994. Fumio Mizutani received his PhD degree from Tokyo Institute of Technology in 1978. Osamu Niwa received his PhD degree from Kyushu University in 1991.
Sensors and Actuators B 130 (2008) 320–325
Surface modification of thin polyion complex film for surface plasmon resonance immunosensor Ryoji Kurita a,∗ , Yoshiki Hirata a , Soichi Yabuki a , Yoshimi Yokota a , Dai Kato a , Yukari Sato a , Fumio Mizutani b , Osamu Niwa a a
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, Japan b University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan Available online 10 August 2007
Abstract A surface plasmon resonance-based immunosensor was developed by modifying the surface of a thin polyion complex film consisting of polyl-lysine and poly-styrenesulfonate. The suppression of non-specific protein adsorption was investigated by changing the blend ratio of the two polymers. The protein adsorption fell to less than 10% that of an unmodified surface when the poly-l-lysine to ratio was 4:6. The binding constant between immobilized B-type natriuretic peptide on the polyion complex film and its antibody was calculated to be 1.1 × 108 (M−1 ), which is a satisfactory level for immuno-affinity on a solid surface. We also obtained stable and reproducible responses without losing the affinity on the polyion complex film. © 2007 Elsevier B.V. All rights reserved. Keywords: Surface plasmon resonance; Immunosensor; Polyion complex film; Protein adsorption
1. Introduction Surface plasmon resonance (SPR) measurement is a surface sensitive analytical technique that has been successfully incorporated into an immunosensor format for the non-labeled assay of various biomolecules [1–5]. The SPR immunosensor also has advantages as regards bedside monitoring and point-of-care testing (POCT) because the method is a rapid, simple, safe and low power technique. However, its major disadvantage is its poor sensitivity to small molecules and poor selectivity resulting from non-specific adsorption. In principle, the sensitivity of the SPR immunosensor depends on the molecular weight of the analytes. Thus, the sensitivity to small molecules is lower than that of other optical methods such as conventional fluorescence detection because small molecule bindings have little effect on the refractive index on a sensing surface. For example, the detection limit of the SPR immunoassay is reported to be around several tens of nM for small peptides [6,7]. In addition, some immobilized antibod-
∗
Corresponding author. Tel.: +81 29 861 6166; fax: +81 29 861 6177. E-mail address: [email protected] (R. Kurita).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.08.007
ies are unable to form stable layers because they are denatured to lose their specific activity compared with that of free antibodies [8]. Very recently, we developed a highly sensitive enzymelabeled SPR immunosensor using an antibody labeled with acetylcholine–esterase, which produces thiol molecules [9]. The detection limit was greatly improved by the thiol accumulation effect in a micro-flow channel, and this proposed immunosensor is one of the most sensitive sensors yet reported based on an SPR detection system. On the other hand, non-specific adsorption on the sensing surface, especially protein fouling [10,11], is a serious problem when measuring trace level biomolecules in real samples using a conventional SPR immunosensor, which detects an SPR angle shift caused by the formation of an immuno-complex on the sensing surface. This is because the SPR angle is increased by the non-specific protein adsorption, which makes it difficult to detect small SPR angle shifts caused by an immuno-reaction with a small amount of analyte. Various kinds of films and modification methods have been studied to suppress the non-specific adsorption that occurs during SPR measurements [12–14], for example, dextran, polyethylene glycol [15], and poly-vinylalcohol [16]. For a highly sensitive and selective SPR immunosensor, it would be better
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2. Experimental
vated the film for 15 min. We then added 1 L of 2 mg/mL BNP (in acetate buffer) to the buffer on the polyion complex film and stirred it for a few seconds with a micropipette, followed by 15 min incubation. After rinsing with acetate buffer, we quenched any unreacted NHS-activated groups by incubation for 5 min with 0.1 M aminoethanol (in acetate buffer). All immobilization reactions were performed at room temperature. The PDMS cover was formed with previously reported procedures [20]. The master pattern was fabricated on a glass wafer by a conventional photolithographic technique with a positive photoresist (PMER P-LA900PM, Tokyo Ohka Kogyo, Japan). Liquid PDMS oligomer (Dow Corning Asia, Japan) and a crosslinking agent were mixed and poured onto the master with the flow channel pattern. After curing the mixture for 60 min at 60 ◦ C, the PDMS layer was removed from the master.
2.1. Reagents
2.3. Measurements using immunosensor
Poly-l-lysine hydrobromide (average M.W. 80,000) was purchased from Sigma (St. Louis, MO). Poly-(sodium 4styrenesulfonate) (average M.W. 70,000) and bovine serum albumin were purchased from Aldrich. 0.1 M phosphate buffer (pH 7.0) and glycine were purchased from Nakalai (Kyoto, Japan). N-hydroxysuccinimide (NHS) and N-ethyl-N (3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) were purchased from Pierce (Rockford, IL). Human B-type natriuretic peptide-32 (BNP) was purchased from Phoenix Pharmaceuticals (Melmont, CA). Mouse monoclonal anti-human B-type natriuretic peptide-32 antibody (anti-BNP antibody) was purchased from ABCAM (Cambridge, UK). Polymer solutions were prepared with Millipore water (Millipore Co.).
Before undertaking any measurements, we peeled the adhesive sheet from the glass plate, and attached the plate to the PDMS cover with the micro-channel. We then connected inlet and outlet tubes (0.12 mm i.d., 0.7 mm o.d., BAS, Japan) to the PDMS cover. We connected the other end of the outlet tube to a syringe, which was equipped with a syringe pump (CMA, Sweden). The sample solution was injected via the sample tube in the suction mode at a flow rate of 2 L/min. First, we introduced a phosphate buffer solution (blank solution) to obtain a stable baseline, then we introduced the sample solution and monitored the SPR angle shift value (θ SPR ). We obtained an SPR curve and measured its minimum reflection angle (SPR angle) using portable SPR equipment (NTT-AT, Japan), which was developed in collaboration with one of the present authors. Index matching oil (n = 1.510, Cargille Laboratories Inc.) was used to obtain optical contact between the glass plate of the immunosensor and the prism on the SPR equipment.
if the membrane on the sensing surface had both a less nonspecific adsorption property and a high density of functional groups for immobilizing antibodies (or antigens) without losing their specific binding activity. In this study, we report the surface modification of an SPR immunosensor with a thin polyion complex film consisting of poly-l-lysine and poly-styrenesulfonate. We optimized the modification of the film and studied the effects of the non-specific adsorption of protein, especially albumin, which is found in large amounts in blood, using SPR and AFM while varying the thickness and blend ratio for the film formation. We also measured the binding constant between antigens and antibodies on the polyion complex film.
2.2. Fabrication of immunosensor with polyion complex film The immunosensor consists of a glass plate (16 mm × 16 mm) with a thin gold film, and a poly-dimethylsiloxane (PDMS) cover with a micro-channel (20 m deep, 2 mm wide). Thin titanium was deposited on the glass plate by using an RF sputtering machine, and gold film was then formed without breaking the vacuum [17]. The total thickness of the gold and titanium film was 50 ± 2 nm. We then sealed the glass plate using a polymeric adhesive sheet with a 3 mm diameter hole. Next, we placed a mixture solution of poly-l-lysine and polystyrenesulfonate on the gold film on the plate, and allowed the plate to dry for one night at room temperature [18]. The polyl-lysine and poly-styrenesulfonate concentrations were varied from 0.62 pg/mm2 to 62 ng/mm2 to obtain films with different thicknesses. We also changed the blend ratio of the polyion complex film from 7:3 to 1:9 by changing the amounts of both solutions. The total volume of solution loaded on each plate was 10 L (1.4 L/mm2 ). When we examined the immuno-affinity on the polyion complex film, we used the following common carbodiimide coupling reaction provided by an EDC/NHS system [19]. We placed 9 L of 10 mM acetate buffer (pH 5.5) containing 0.4 mg/mL EDC and 1.1 mg/mL NHS on the polyion complex film, and acti-
3. Results and discussions 3.1. Polyion complex film formation for SPR measurement Fig. 1(a) and (b) shows the respective variations in the SPR curve shape and the SPR angle for distilled water when we varied the concentrations of the loaded polyion complex solution for film formation. The SPR angles were around 67.5◦ , and narrow SPR curves were observed when the polymer concentration was less than 1 ng/mm2 . However, the SPR angle became large and the SPR curve broadened when the concentrations were greater than 1 ng/mm2 . Fig. 2 shows AFM images of the polyion complex film on the gold surface. The gold surface was covered with the film, and flat hill-like structures were observed in the AFM images. When the polymer concentration was increased, many grains were observed and this caused the SPR angle increase. This indicates that a solution with a high polyion complex concentration is inappropriate as regards forming a film for SPR measurement because a high concentration solution tends to form many grains rather than a thin film on the gold surface. AFM observation revealed that the film
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Fig. 1. (a) SPR curves when the concentrations of the loaded polyion complex solution on a gold surface were 0.62, 6.2, 62 ng/mm2 , respectively. (b) Variation in SPR angle for distilled water when the polyion complex solution was changed over a wide range.
Fig. 2. AFM images (10 m × 10 m) of a gold surface modified with the polyion complex film when the polyion complex solutions were varied. (a) 0.62 ng/mm2 , (b) 2.48 ng/mm2 , (c) 3.72 ng/mm2 . The height profiles are given beneath each AFM image.
thickness was around 5 nm when the polymer concentration was around 1 ng/mm2 . Based on the AFM observation and the SPR angles in Fig. 1, we chose a film formed with a concentration of 0.62 ng/mm2 . This is because a thin polymer layer is advantageous for SPR measurement since the intensity of an evanescent field decreases with distance from the gold surface. The polymer was much thinner than previously reported modified polymers, other than those where the self-assembly technique was used.
adsorption was observed when the film composition was slightly poly-styrenesulfonate rich and weakly anionic charged. This could suggest that electrostatic repulsion contributes slightly to the rejection of albumin, since it is an anionic protein in a neutral solution.
3.2. Non-specific adsorption properties of polyion complex film Fig. 3 shows the variation in the SPR angle shift value (θ SPR ) for 1 mg/mL albumin solution injection when we varied the blend ratio of the polyion complexes used for film formation. The θ SPR was almost the same as that of an unmodified gold surface for very different blend ratios. However, protein fouling was suppressed when the poly-l-lysine to poly-styrenesulfonate blend ratio was between 5:5 and 3:7. Non-specific adsorption was well suppressed when the blend ratio was 4:6, and was less than 10% that of bare gold film. Many researchers have reported various non-protein adsorption polymers, which are neutral and hydrophilic. Our polyion complex film also suppresses protein adsorption when the polyanion and polycation blend content was almost the same. However, the minimum
Fig. 3. Variation in the non-specific adsorption for 1 mg/mL of albumin when the poly-l-lysine to poly-styrenesulfonate blend ratio is varied. Albumin solution was injected for 5 min at a flow rate of 2 L/min.
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Fig. 4. (a) SPR sensorgrams when 1–100 g/mL of anti-BNP antibody solutions was introduced onto the polyion complex film modified with BNP. (b) Scatchard plot analysis of bound anti-BNP antibody against bound/free anti-BNP antibody obtained from Fig. 4(a).
Based on the θ SPR , about 90 pg/mm2 of albumin was adsorbed on the polyion complex film when the blend ratio was 4:6 since a 1◦ SPR angle shift is reported to correspond to a protein adsorption of 10 ng/mm2 [21,22]. The 90 pg/mm2 albumin adsorption is almost equivalent to the amount of non-specific albumin adsorption on a commercially available dextran film, which is one of the most widely used films for SPR measurement [23]. Although the amount of adsorbed albumin reported in this paper is slightly larger than that of synthesized polymers designed to suppress non-specific protein adsorption such as ␣-acetal--mercapto-poly-ethyleneglycol (24 pg/mm2 ) [24] and poly-sulfobetaine methacrylate (38 pg/mm2 ) [25], we are able to obtain the film with a simple and rapid process solely by mixing two common commercially available polymers. Furthermore, it is difficult to modify poly-ethylene glycol with an antigen or antibody because it contains no functional groups, such as amino or carboxyl groups, which are commonly used to immobilize antibodies or antigens. In contrast, our polyion complex film is advantageous for an SPR immunosensor since the film has both amino and carboxyl groups. In this study, we have focused particularly on the albumin rejection among proteins for future POCT application because blood samples contain a large amount of albumin. The polyion complex film may also be effective for rejecting other proteins since the charge in the
polymer film can be tuned by changing the poly-l-lysine to poly-styrenesulfonate blend ratio, however, this requires further investigation. 3.3. Immuno-affinity on polyion complex film An immunosensor with a low detection limit can be obtained by increasing the amount of antibody or antigen without losing its activity in addition to low non-specific adsorption. Therefore, in the next step, we examined the binding constant of immunoaffinity on the polyion complex film because it is known that antibodies or antigens immobilized on a solid surface sometimes lose their specific activity. Fig. 4(a) shows SPR sensorgrams when we injected 1 to 100 g/mL of anti-BNP antibody solution for 10 min into an immunosensor prepared by immobilizing BNP on the polyion complex film. We observed a large θ SPR as the antibody concentration increased. We also obtained a Scatchard plot as shown in Fig. 4(b) from the SPR sensorgrams of 5–100 g/mL antibody solutions in Fig. 4(a) except with 1 and 2 g/mL antibody injections. This exception is because the two SPR sensorgrams did not remain in a steady state during the 10 min antibody injection, i.e. the antigen–antibody reaction on the polyion complex film did not reach equilibrium. The binding constant of the immuno-reaction was estimated to be
Fig. 5. (a) SPR sensorgrams for 10 g/mL anti-BNP antibody solutions containing 0, 0.04, 0.4, 4, 40, 400 g/mL BNP on polyion complex film modified with BNP. (1) Baseline for blank buffer. (2) Injection of a mixture solution of BNP and anti-BNP antibody. (3) Injection of buffer and θ SPR measurement. (4) Regeneration with glycine–HCl. (b) Calibration curve for BNP. Y-axis is θ SPR at 3rd step obtained from Fig. 5(a).
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Fig. 6 shows the SPR sensorgrams we obtained when we injected a mixture solution of 10 g/mL anti-BNP antibody and 400 ng/mL BNP into the immunosensor five times after regeneration with glycine–HCl buffer (pH 2.0). The heights and shapes of the SPR sensorgrams were almost the same and we observed no reduction in the response. The relative standard deviation in the θ SPR for the five measurements was 3.4%. We were able to obtain stable and reproducible responses using our immunosensor with the thin polyion complex film. 4. Conclusion
Fig. 6. SPR sensorgrams when a mixture solution of BNP and anti-BNP antibody was injected repeatedly after regeneration with glycine–HCl buffer. Filled and open bars indicate the injection of the mixture solution and glycine–HCl buffer, respectively.
1.1 × 108 (M−1 ). This value is a satisfactory level for immunoaffinity on a solid surface [26,27]. We believe this to be because polyion complex film has a high biocompatibility without denaturation. Fig. 5(a) shows SPR sensorgrams for 10 g/mL anti-BNP antibody solutions on the BNP immobilized polyion complex film surface after reacting with sample BNP with concentrations ranging from 0 to 400 g/mL. We introduced a blank buffer solution for 1 min onto the BNP modified polyion complex film, and obtained a stable baseline as the first step (1). Then we introduced a mixture solution of BNP and anti-BNP antibody onto the sensing surface for 5 min, after 20 min of incubation in a microcuvetto as the second step (2). In this step, the reacted antibodies are not bound to the BNP on the polyion complex film. In contrast, only unreacted anti-BNP antibodies bound to the BNP on the film and cause an SPR angle increase. In the third step, we again introduced a buffer solution for 3 min and measured the θ SPR (3). Finally, we regenerated the film with an injection of glycine–HCl buffer (pH 2.0) for 3 min (4). Fig. 5(b) shows the calibration curve for BNP obtained from SPR sensorgrams in Fig. 5(a). We observed a large θ SPR when the BNP concentration was low. However, the θ SPR decreased as the BNP concentration increased. This is because the unreacted antibody decreased as the sample BNP concentration increased, so the amount of bounded antibody on the polyion complex film decreased. The detection limit for the BNP concentration with our immunosensor was around a few tens of ng/mL (about 1 nM). This value is about one order of magnitude better than that obtained for previously reported non-labeled SPR immunosensors for small peptides [6,7]. A non-labeled SPR immunosensor usually detects SPR angle shifts caused by small analytes binding with the antibody modified sensing surface. Our immunosensor is advantageous in terms of improving the sensitivity since we measured unreacted antibodies whose molecular weight (150 kDa) was larger that that of BNP (3465). This was achieved by the highly suppressing property of the non-specific adsorption of reacted antibodies on the polyion complex film.
This paper describes the modification of a gold sensing surface for an SPR-based immunosensor with a thin polyion complex film consisting of poly-l-lysine and polystyrenesulfonate. The film suppresses non-specific protein adsorption when the film thickness and the blend ratio of the two polymers are optimized. Biomolecules such as small peptides can also be immobilized on our film with a relatively high density without losing binding activity. The polyion complex film is beneficial for modifying the sensing surface of a non-labeled direct SPR immunosensor. Although we describe only BNP monitoring, the film could be applied to various biomolecules by changing both the antigen on the film and its antibody. References [1] V. Kanda, J.K. Kariuki, D.J. Harrison, M.T. McDermott, Label-free reading of microarray-based immunoassays with surface plasmon resonance imaging, Anal. Chem. 76 (2004) 7257–7262. [2] B. Fitzpatrick, R. O’Kennedy, The development and application of a surface plasmon resonance-based inhibition immunoassay for the determination of warfarin in plasma ultrafiltrate, J. Immunol. Methods 291 (2004) 11–25. [3] T. Akimoto, K. Ikebukuro, I. Karube, A surface plasmon resonance probe with a novel integrated reference sensor surface, Biosens. Bioelectron. 18 (2003) 1447–1453. [4] K.V. Gobi, C. Kataoka, N. Miura, Surface plasmon resonance detection of endocrine disruptors using immunoprobes based on self-assembled monolayers, Sens. Actuators B: Chem. 108 (2005) 784–790. [5] T. Uda, K. Inoue, T. Nishimura, E. Hifumi, K. Shimizu, N. Egashira, Simultaneous detection of two antigens by surface plasmon resonance sensor, Electrochemistry 69 (2001) 976–979. [6] P. Gomes, D. Andreu, Direct kinetic assay of interactions between small peptides and immobilized antibodies using a surface plasmon resonance biosensor, J. Immunol. Methods 259 (2002) 217–230. [7] R.P.H. Kooyman, H. Kolkman, J. Van Gent, J. Greve, Surface plasmon resonance immunosensors: sensitivity considerations, Anal. Chim. Acta 213 (1988) 35–45. [8] B. Oh, W. Lee, Y. Kim, W.H. Lee, J. Choi, Surface plasmon resonance immunosensor using self-assembled protein G for the detection of salmonella paratyphi, J. Biotechnol. 111 (2004) 1–8. [9] R. Kurita, Y. Yokota, Y. Sato, F. Mizutani, O. Niwa, On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system, Anal. Chem. 78 (2006) 5525–5531. [10] R. Kurita, H. Tabei, Y. Iwasaki, K. Hayashi, K. Sunagawa, O. Niwa, Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane, Biosens. Bioelectron. 20 (2004) 518–523. [11] R. Kurita, N. Yabumoto, O. Niwa, Miniaturized one-chip electrochemical sensing device integrated with a dialysis membrane and double thin-layer flow channels for measuring blood samples, Biosens. Bioelectron. 21 (2006) 1649–1653.
R. Kurita et al. / Sensors and Actuators B 130 (2008) 320–325 [12] V. Silin, H. Weetall, D.J. Vanderah, SPR studies of the nonspecific adsorption kinetics of human IgG and BSA on gold surfaces modified by self-assembled monolayers (SAMs), J. Colloid Interface Sci. 185 (1997) 94–103. [13] M. Mrksich, G.B. Sigal, G.M. Whitesides, Surface-plasmon resonance permits in-situ measurement of protein adsorption on self-assembled monolayers of alkanethiolates on gold, Langmuir 11 (1995) 4383–4385. [14] S. Toyama, A. Shoji, Y. Yoshida, S. Yamauchi, Y. Ikariyama, Surface design of SPR-based immunosensor for the effective binding of antigen or antibody in the evanescent field using mixed polymer matrix, Sens. Actuators B: Chem. 52 (1998) 65–71. [15] J.M. Brockman, A.G. Frutos, R.M. Corn, A multistep chemical modification procedure to create DNA arrays on gold surfaces for the study of protein-DNA interactions with surface plasmon resonance imaging, J. Am. Chem. Soc. 121 (1999) 8044–8051. [16] D.A. Barrett, M.S. Hartshorne, M.A. Hussain, P.N. Shaw, M.C. Davis, Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance, Anal. Chem. 73 (2001) 5232–5239. [17] R. Kurita, H. Tabei, Z. Liu, T. Horiuchi, O. Niwa, Fabrication and electroproperties of an interdigitated array electrode in a microfabricated wall-jet cell, Sens. Actuators B: Chem. 71 (2000) 82–89. [18] S. Yabuki, F. Mizutani, Y. Hirata, Preparation of D-amino acid oxidaseimmobilized polyion complex membranes, Sens. Actuators B: Chem. 76 (2001) 142–146. [19] M.R. Lewis, A. Raubitschek, J.E. Shively, A facile water-soluble method for modification of proteins with DOTA: use of elevated-temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled, Bioconjug. Chem. 5 (1994) 565–576. [20] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Rapid prototyping of microfluidic systems in poly(dimethylsiloxane), Anal. Chem. 70 (1998) 4974–4984. [21] L.G. Fagerstam, A. Frostellkarlsson, R. Karlsson, B. Persson, I. Ronnberg, Biospecific interaction analysis using surface-plasmon resonance detection applied to kinetic, binding-site and concentration analysis, J. Chromatogr. 597 (1992) 397–410. [22] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitativedetermination of surface concentration of protein with surface-plasmon resonance using radiolabeled proteins, J. Colloid Interface Sci. 143 (1991) 513–526.
325
[23] R.A. Frazier, G. Matthijs, M.C. Davies, C.J. Roberts, E. Schacht, S.J.B. Tendler, Characterization of protein-resistant dextran monolayers, Biomaterials 21 (2000) 957–966. [24] K. Uchida, H. Otsuka, M. Kaneko, K. Kataoka, Y. Nagasaki, A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: the substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption, Anal. Chem. 77 (2005) 1075–1080. [25] Y. Chang, S. Chen, Z. Zhang, S. Jiang, Highly protein-resistant coatings from well-defined diblock copolymers containing sulfobetaines, Langmuir 22 (2006) 2222–2226. [26] M.H.F. Meyer, M. Hartmann, M. Keusgen, SPR-based immunosensor for the CRP detection—a new method to detect a well known protein, Biosens. Bioelectron. 21 (2006) 1987–1990. [27] Y. Kikuchi, S. Uno, M. Nanami, Y. Yoshimura, S. Iida, N. Fukushima, M. Tsuchiya, Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance, J. Biosci. Bioeng. 100 (2005) 311–317.
Biographies Ryoji Kurita received his PhD degree from Kyushu University in 2004. Yoshiki Hirata received his PhD degree from Tokyo Institute of Technology in 1990. Soichi Yabuki received his PhD degree from Tokyo Institute of Technology in 1989. Yoshimi Yokota received her MS degree from Kyoto University in 1988. Dai Kato received his PhD degree from Kumamoto University in 2003. Yukari Sato received her PhD degree from Hokkaido University in 1994. Fumio Mizutani received his PhD degree from Tokyo Institute of Technology in 1978. Osamu Niwa received his PhD degree from Kyushu University in 1991.