Polyelectrolyte Multilayers-Modified Polystyrene Plate Improves

Jul 16, 2009 - Department of Applied Chemistry, Graduate School of Engineering, Osaka University, and 21st Century COE for “Center for Integrated Ce...
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Anal. Chem. 2009, 81, 6923–6928

Polyelectrolyte Multilayers-Modified Polystyrene Plate Improves Conventional Immunoassay: Full Covering of the Blocking Reagent Heyun Shen,†,‡ Junji Watanabe,†,‡ and Mitsuru Akashi*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, and 21st Century COE for “Center for Integrated Cell and Tissue Regulation”, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan In this study, we fabricated polyelectrolyte multilayers (PEMs) on a polystyrene (PS) plate by a simple and novel alternate drop coating process (Acta Biomaterialia 2008, 4, 1255-1262), leading to the construction of a functional platform for improving conventional enzyme-linked immunosorbent assay (ELISA) systems. Poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) were used as cationic and anionic polyelectrolytes, and then positively or negatively charged surfaces were obtained on the PEMs. The PDDA/PSS PEMs on the PS plate had the following favorable characteristics. On the positive PEMs, the coverage of the blocking reagent (ovalbumin from egg white: OVA) was over 100% by electrostatic interaction between the protein and PEMs, hence, nonspecific adsorption from the secondary antibody was efficiently suppressed. Moreover, the positive PEMs showed higher sensitivity on the ELISA than the negative PEMs, including the PS plate. Regularly oscillating behavior for sensitivity (specific signal-to-noise ratio) was observed on 1-10-step assemblies. The calibration curves for antigen detection on the positive PEMs had wide range of concentration from 0.002 to 5 µg/mL, and largest change in signal as compared with the negative PEMs and the PS plate. In summary, we discovered that positive PEMs possessed excellent performance for ELISA systems, and PEMs could easily improve the immunoassay with a convenient process and diverse substrates. The construction of functional protein adsorption films is an important field of research in biotechnology. Control over the surface properties of ultrathin films, such as wettability and adsorption behavior, are important in a wide variety of applications, including surface self-cleaning, antiadhesives, biosensing, and separation. Polyelectrolyte multilayers (PEMs) have shown excellent potential in various biomaterial applications since they were first discovered by Decher in the early 1990s.1,2 Using given oppositely charged polyelectrolytes, one can easily prepare PEMs by controlling their structure at the nanometer level using layer* To whom correspondence should be addressed. E-mail: akashi@ chem.eng.osaka-u.ac.jp. Phone: +81-6-6879-7356. Fax: +81-6-6879-7359. † Department of Applied Chemistry. ‡ Center for Integrated Cell and Tissue Regulation. (1) Decher, G.; Hong, J. D. Macromol. Chem., Macromol. Symp. 1991, 46, 321–327. (2) Decher, G.; Schmitt, J. Thin Solid Films 1992, 831, 210–211. 10.1021/ac900985x CCC: $40.75  2009 American Chemical Society Published on Web 07/16/2009

by-layer (LbL) technology.3 The products from the PEMs were robust under ambient and physiological conditions; hence, this process is useful for surface modifications, which are extraordinarily advanced for biomedical applications.4 For instance, a number of studies have demonstrated that PEMs exhibit versatile functions with regard to protein adsorption.5-10 The driving forces for protein adsorption on the PEMs were considered to be electrostatic forces, hydrogen bonding, and hydrophobic interactions. Based on the diverse behavior of protein adsorption, we fabricated a heterofunctional interface from differently charged PEMs on both sides of a substrate by a novel alternate drop coating process,11 which could simultaneously regulate the amount of protein adsorption on each side of the substrate by electrostatic forces.12 Moreover, the heterofunctional interface could achieve selectively different charged protein adsorption on each side of the substrate in the protein mixture.13 Taking this into account, we considered that the PEMs could also control the protein adsorption during each process in the enzyme-linked immunosorbent assay (ELISA) system, thus leading to an improved immunoassay. Caruso et al. investigated the behavior of immunoglobulin (IgG) adsorption on and/or in poly(sodium 4-styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) PEMs coated polystyrene (PS) particles, and observed that the IgG retained its natural conformation; hence, the PEMs coated PS particles could contribute to immunoassay applications.14-16 Recently, PEMs (3) Decher, G. Science 1997, 277, 1232–1237. (4) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203–3224. (5) Ladam, G.; Schaaf, P.; Decher, G.; Voegel, J. C.; Cuisinier, F. J. G. Biomol. Eng. 2002, 19, 273–280. (6) Schwinte, P.; Ball, V.; Szalontai, B.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Biomacromolecules 2002, 3, 1135–1143. (7) Mu ¨ ller, M.; Rieser, T.; Dubin, P.; Lunkwitz, K. Macromol. Rapid Commun. 2001, 22, 390–395. (8) Izumrudov, V. A.; Kharlampieva, E.; Sukhishvili, S. A. Biomacromolecules 2005, 6, 1782–1788. (9) Salloum, D. S.; Schlenoff, J. B. Biomacromolecules 2004, 5, 1089–1096. (10) Gergely, C.; Bahi, S.; Szalontai, B.; Flores, H.; Schaaf, P.; Voegel, J. C.; Cuisinier, F. J. G. Langmuir 2004, 20, 5575–5582. (11) Watanabe, J.; Shen, H. Y.; Akashi, M. Acta Biomater. 2008, 4, 1255–1262. (12) Watanabe, J.; Shen, H. Y.; Akashi, M. J. Mater. Sci. Mater. Med. 2009, 20, 759–765. (13) Shen, H. Y.; Watanabe, J.; Akashi, M. Polym. J. 2009, 41, 486–491. (14) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 4559–4565. (15) Caruso, F.; Mo ¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039–6046. (16) Yang, W. J.; Trau, D.; Renneberg, R.; Yu, N. T.; Caruso, F. J. Colloid Interface Sci. 2001, 234, 356–362.

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were used in advanced immunoassay studies.17-19 Poly(ethyleneimine) (PEI)/poly(acrylic acid) (PAA) PEMs were coated onto poly(dimethylsiloxane) (PDMS) with subsequent cross-linking to create long-term stability on the interface for protein detection.17 PAH/PAA PEMs have been modified on porous alumina membranes18 and PS particles,19 demonstrating that the PAA layer could resist nonspecific adsorption and allow for the covalent immobilization of proteins. However, the use of PEMs in the conventional ELISA system, including a stepwise adsorption process of the primary antibody, blocking regent, antigen, and secondary antibody, has not yet been demonstrated in detail. A number of researchers have created high performance devices in order to develop highly sensitive ELISA systems. Nagasaki et al. coimmobilized poly(ethylene glycol) (PEG) and antibodies on a substrate to evaluate the sandwich immunoassay system. Interestingly, when a mixture of different molecular weights of PEG brush was immobilized, the substrate not only suppressed nonspecific adsorption, but also enhanced the corresponding epitope.20 Porter et al. reported that the Fab-SH fragment could be immobilized onto a gold substrate by a thiolate bond. The higher epitope density was achieved relative to immobilize whole molecule of antibody on the surface, which improved the antigen recognition on the surface.21 Serizawa et al. demonstrated that the amount of protein A adsorption on the poly(methyl methacrylate) (PMMA) stereocomplex film was greater than that of single-component of PMMA films, resulting in higher antibody immobilization on the stereocomplex while exposing the epitope moiety.22 However, it should be noted that the above strategies cannot easily handle extensive applications due to the complicated processes and expensive cost. Therefore, we considered that the PEMs for simple processes should be developed in various immunoassay devices with high sensitivity. In this study, we discovered that the PEMs could improve the conventional ELISA system in comparison with the conventional PS plate, due to the significant blocking reagent adsorption by electrostatic interactions between the PEMs and ovalbumin from egg white (OVA). Polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and PSS were selected as cationic and anionic polyelectrolytes. These polymer combinations have been used to fabricate PEMs by many researchers.9,23,24 One to ten stepwise assemblies of PDDA/PSS PEMs were previously deposited on PS plates by an alternate drop coating process.11 We demonstrated that this alternate drop coating process was similar to the conventional alternate adsorption process, including the thickness and biofunctions of the PEMs from our previous study. The adsorbed antibody and blocking reagent (OVA) were investigated. On the positive PEMs, the amount of primary antibody adsorption was less and the OVA adsorption was higher than adsorption on (17) Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H. Anal. Chem. 2008, 80, 1529–1535. (18) Dai, J. H.; Bake, G. L.; Bruening, M. L. Anal. Chem. 2006, 78, 135–140. (19) Derveaux, S.; Stubbe, B. G.; Roelant, C.; Leblans, M.; De Geest, B. G.; Demeester, J.; De Smedt, S. C. Anal. Chem. 2008, 80, 85–94. (20) Nagasaki, Y.; Kobayashi, H.; Katsuyama, Y.; Jomura, T.; Sakura, T. J. Colloid Interface Sci. 2007, 309, 524–530. (21) O’Brien, J. C.; Jones, V. W.; Porter, M. D. Anal. Chem. 2000, 72, 703–710. (22) Serizawa, T.; Nagasaka, Y.; Matsuno, H.; Shimoyama, M.; Kurita, K. Bioconjugate Chem. 2007, 18, 355–362. (23) Liu, H. Y.; Hu, N. F. J. Phys. Chem. B 2005, 109, 10464–10473. (24) Roy, X.; Sarazin, P.; Favis, B. D. Adv. Mater. 2006, 18, 1015–1019.

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Figure 1. Chemical structures of the polyelectrolytes used in this study.

the negative PEMs and the PS plate. Moreover, on the positive PEMs, the OVA coverage was over 100% which might be bilayer adsorption; hence, it efficiently suppressed the nonspecific adsorption from the secondary antibody, leading to a higher sensitivity for antigen detection. Therefore, higher sensitivity could be achieved on the PEMs surface in the ELISA system. From the present study, we demonstrated that the PEMs could contribute to the enhanced sensitivity of the conventional ELISA system due to its significant blocking effect. MATERIALS AND METHODS Materials. The polyelectrolytes used in this study are shown in Figure 1. Poly(diallyldimethylammonium chloride) (PDDA, no. 17338; Polyscience Inc., PA; Mw ) 2.4 × 105 g/mol) and poly(sodium 4-styrenesulfonate) (PSS, no. 561959; Aldrich, MO; Mw ) 2.0 × 105 g/mol) were used as strong polyelectrolytes. Rabbit antimouse IgG (M7023), mouse IgG (I5381), goat antimouse IgG- horseradish peroxidase conjugate (goat antimouse IgG-HRP; A4416), and ovalbumin from chicken egg white (OVA, A5503) were purchased from SIGMA, MO. Tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl; Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as a buffer solution. All chemicals were used without further purification. Ultrapure water was used throughout this experiment. Fabrication of PEMs in PS Microplate Wells. The polyelectrolyte solutionss PDDA and PSSswere separately dissolved in 50 mmol/L tris-HCl (pH 7.4), and the concentration of each solution was adjusted to 0.2 mg/mL. The ionic strength of each solution was then adjusted to 0.15 mol/L using NaCl. At first, we dropped 120 µL of PDDA aqueous solution in the wells of a PS microplate (48 or 96 wells) for 1 min at room temperature; subsequently, the plate was rinsed with 1 mmol/L tris-HCl (pH 7.4) for 20 s and then removed. Next, 120 µL of PSS aqueous solution was dropped as described above. This alternate drop coating process was repeated for a given number of cycles for the preparation of the PEMs. Determination of Protein Adsorption. The primary antibody (rabbit antimouse IgG; 31 µg/mL in 50 mmol/L tris-HCl; pH 7.4) was adsorbed onto the PEMs (48 wells) at 4 °C overnight. Thereafter, the plates were rinsed with 1 mmol/L tris-HCl, and then the adsorbed proteins were removed by 1 wt % of n-sodium dodecyl sulfate (SDS; Wako Pure Chemical Industries, Ltd.). The recovered protein from the substrates was evaluated using a Micro BCA kit (no. 23235, Pierce, IL) at 570 nm by a multiwell plate reader (model 680, BIO RAD Co. Ltd., CA). OVA (1 mg/mL; tris-HCl, 50 mmol/L; pH 7.4) was adsorbed onto the PEMs (48 wells) at 37 °C for 1 h. Thereafter, the process

Figure 2. Amount of primary antibody adsorption on the PS plate and PDDA/PSS PEMs. The odd and even steps represent the PDDA and PSS layers, respectively. (n ) 3).

Figure 4. UV-vis absorbance of the signal (S’) and noise (N) of the secondary antibody (a), UV-vis absorbance of specific signal (SdS’sN) (b), and the specific signal (S) to noise (N) ratio (c) on the PS plate and each layer of the PDDA/PSS PEMs. The odd and even steps represent the PDDA and PSS layers, respectively. (n ) 5).

Figure 3. Amount of OVA adsorption on the PS plate and PDDA/ PSS PEMs. The odd and even steps represent the PDDA and PSS layers, respectively. (n ) 3).

was repeated in the same manner as mentioned above to determinate the OVA adsorption. ELISA Protocol on the PEMs. The primary antibody (31 µg/ mL; 100 µL/well) was adsorbed onto PEMs (96 wells) at 4 °C overnight to facilitate antigen detection by ELISA. Next, OVA (1 mg/mL; 200 µL/well) was adsorbed onto each substrate at 37 °C for 1 h. Mouse IgG and goat antimouse IgG-HRP were used as the antigen and secondary antibody, respectively (100 µL/well; tris-HCl 50 mmol/L with 0.1 wt % OVA; pH 7.4). The concentration of antigen was 2 and 4 µg/mL on the positive PEMs (and PS) and negative PEMs, respectively. On the positive PEMs and PS plate, the optimized concentration of secondary antibody was 0.5 µg/mL, whereas the negative PEMs was 1 µg/mL. The antigenantibody reaction was carried out at 37 °C for 1 h. After rinsing with 1 mmol/L tris-HCl, tetramethylbenzidine (ML-1120T, Sumitomo Bakelite Co. Ltd., Tokyo, Japan) was added to each plate for the HRP, and then the absorbance of the solution was measured at 450 nm by the multiwell plate reader. For the calibration curve estimation, the concentration of antigen ranged from 0.002 to 5 µg/mL, and the concentration of the secondary antibody was the optimized concentration for each substrate as mentioned above.

Figure 5. UV-vis absorbance of the specific signal of the secondary antibody with a change in the antigen values on the PS plate (square plots), (PDDA/PSS)3(triangle plots), and (PDDA/PSS)3PDDA (dot plots) PEMs. (n ) 5).

RESULTS AND DISCUSSION Determination of Primary Antibody and Blocking Reagent Adsorption. We reported that PDDA/PSS PEMs could regulate bovine serum albumin (BSA) adsorption by electrostatic interactions between the PEMs and the protein, and the interaction could be inhibited by addition of counterions, such as sodium chloride.11 Moreover, differently charged PDDA/PSS PEMs were precisely fabricated on both sides of the substrate by a novel alternate drop coating process, creating different amounts of protein adsorption12 and selective protein adsorption from a mixture on both sides of the substrate by electrostatic interaction between the protein and PEMs surface.13 Therefore, we considered that the PDDA/PSS PEMs could regulate different kinds of protein adsorption in the Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Table 1. Results of the Primary Antibody and OVA Adsorption and the Ratio of Molecules IgG coveragea(%)

OVA coverageb(%)

surface

primary IgG (µg/cm2)

end-on

side-on

OVA (µg/cm2)

end-on

side-on

ratio of moleculesc (OVA/IgG)

PS plate (PDDA/PSS)3 (PDDA/PSS)3PDDA

0.22 ± 0.03 0.46 ± 0.03 0.22 ± 0.02

12 25 12

81 170 81

0.18 ± 0.06 0.11 ± 0.03 0.52 ± 0.05

42 26 123

66 40 192

2.7 0.6 6.9

a The dimensions of the IgG are 14.5 × 8.5 × 4 nm.28 When one assumes full monolayer coverage, the amount of IgG adsorption was 1.85 and 0.27 µg /cm2 in the end-on and side-on positions, respectively. b The dimensions of OVA are 7 × 5 × 4.5 nm.29 When one assumes full monolayer coverage, the amount of OVA adsorption was 0.42 and 0.27 µg/cm2 in the end-on and side-on positions, respectively. c The molecular weights of IgG and OVA were 150 kDa and 45 kDa, respectively.

ELISA system, and may contribute to a higher sensitivity of the immunoassay. First, we confirmed the primary antibody adsorption on the PS plate, and the 1-10-step assemblies of PDDA/PSS PEMs. As shown in Figure 2, the amount of IgG adsorption was 0.34-0.44 µg/cm2 on the negative PEMs (PSS layers), whereas the positive PEMs (PDDA layers) and PS plate were 0.20-0.28 and 0.21 µg/cm2, respectively. The surface charges of PDDA/PSS PEMs on the PS particles have been estimated by Caruso et al.25 In this study, we estimated the surface charges of PS plate and PDDA/PSS PEMs by a cationic dye (methylene blue) staining. The absorbance of negative PEMs was higher than that of the PS plate and the positive PEMs after staining by electrostatic interaction (Supporting Information Figure S-1), introducing the absorbance oscillation on the 0-10-step PEMs. This result cleared that the surface of PDDA and PSS layer enriched cationic and anionic charge, respectively. Generally, IgG exhibits a neutral isoelectric point (pI 6.4-9.0).26 Therefore, the electrostatic interactions between the whole antibody molecule and the PEMs were fairly reduced, as compared to the case of BSA (pI 4.9). Figure 3 shows the amount of blocking reagent, OVA, adsorbed onto the PS plate and each step of the assembly of PEMs. The amount of OVA adsorption on the positive PEMs was 0.37-0.60 µg/cm2, and the surface coverage was close to or over 100% which might be bilayer adsorption. On the other hand, 0.11-0.27 and 0.18 µg/cm2 of OVA was adsorbed onto the negative PEMs and PS plate, respectively. The pI of OVA is 4.6, close to BSA, hence in the tris-HCl buffer solution (pH 7.4), OVA was anionically charged. In addition, we investigated the surface wettability of PS plate and each layer of PEMs by static contact angle with ultrapure water droplet (Supporting Information Figure S-2). The contact angle of PS plate was 73°, and that gradually changed to hydrophilic due to the stepwise assembly of PDDA/PSS PEMs. Over the 6-step assembly, the static contact angle reached approximately 30-40° which was regardless of PDDA or PSS layers. Therefore, we considered that electrostatic interaction was the dominant driving force for the protein adsorption on the PDDA/PSS PEMs rather than hydrophobic interaction, resulting in greater amount of OVA adsorption on the positive PEMs than on the negative PEMs and PS plate. In general, the surface properties of PEMs were precisely regulated by outermost layer of polyelectrolyte over 6-step (25) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo ¨hwald, H. Macromolecules 1999, 32, 2317–2328. (26) Li, G.; Stewart, R.; Conlan, B.; Gilbert, A.; Roeth, P.; Nair, H. Vox Sang. 2002, 83, 332–338.

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Table 2. Optimized Concentration of Antigen and Secondary Antibody on Each Substrate surface

antigen (µg/mL)

secondary antibody (µg/mL)

PS plate (PDDA/PSS)3 (PDDA/PSS)3DDA

2 4 2

0.5 1 0.5

Table 3. Chang in Signal on the Antigen Detection signal change in the different concentration rangea (abs) surface

slope of the linea

0.002-0.02

0.002-5 (µg/mL)

PS plate (PDDA/PSS)3 (PDDA/PSS)3PDDA

0.07 0.02 0.13

0.12 0.06 0.33

0.53 0.16 1.03

a

The data was calculated from Figure 5.

assembly.27 Therefore, based on the amount of primary antibody and OVA adsorption, we estimated the surface coverage of protein adsorption and the ratio of protein molecules on the PS plate, (PDDA/PSS)3 (6-step assembly; PSS was outermost layer) and (PDDA/PSS)3PDDA (7-step assembly; PDDA was outermost layer) PEMs, respectively (Table 1). It should be noted that the coverage of OVA adsorption on (PDDA/PSS)3PDDA exceeded 100%, and was unrelated to the direction of the adsorbed OVA. Moreover, the ratios of molecules (OVA/IgG) showed a large difference for each substrate. The ratio of molecules for (PDDA/PSS)3PDDA was 6.9, whereas the (PDDA/PSS)3 and PS plate were 0.6 and 2.7, respectively, resulting in the ratio value for (PDDA/PSS)3PDDA that was 11.5 times and 2.5 times higher than that of (PDDA/PSS)3 and the PS plate, respectively. Taking this result into account, we conclude that (PDDA/ PSS)3PDDA PEMs have excellent potential to improve the conventional ELISA system due to the efficient blocking effect per adsorbed primary antibody. Improvement of Detection Sensitivity on Cationic PEMs. In this study, we aimed to improve the conventional ELISA system on the PS plate using PDDA/PSS PEMs. First, we investigated the sensitivity of antigen detection (specific signal-to-noise ratio; S/N) of the PEMs and PS plate in order to estimate the PEMs (27) Sakaguchi, H.; Serizawa, T.; Akashi, M. Chem. Lett. 2003, 32, 174–175. (28) Silverton, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140–5144. (29) Kamilya, T.; Pal, P.; Talapatra, G. B. J. Phys. Chem. B 2007, 111, 1199– 1205.

Figure 6. Speculation of the signal (S’) and noise (N) of the secondary antibody on the PS plate, (PDDA/PSS)3, and (PDDA/PSS)3PDDA, respectively.

performance in the ELISA system. The concentrations of the antigen and secondary antibody were optimized to attain the maximum S/N value on the PS plate, (PDDA/PSS)3, and (PDDA/ PSS)3PDDA, respectively (Table 2). The antigen and antibody optimizations were carried out in range of 0.25-4 µg/mL and 0.5-8 µg/mL, respectively. Over 6-step PEMs, the physical characteristics of the PEMs surface were precisely regulated by the outermost layer of polyelectrolyte. Moreover, the primary antibody and OVA adsorption on (PDDA/PSS)3 and (PDDA/ PSS)3PDDA were relatively stable; hence, we selected them as typical negative and positive PEMs, and the results of the optimized concentration were used as negative and positive PEMs of the 1-10-step assemblies. As shown in Figure 4, we can observe regularly oscillating behaviors in the signal and noise absorbance, and the S/N ratio from 2- to 10-step assembly. However, the values of specific signal (S; the signal of antigensecondary antibody reaction) by subtracting the noise from the signal on the positive PEMs were larger than that of the negative PEMs and PS plate, since the noise (nonspecific adsorption of secondary antibody) was efficiently suppressed by the significant blocking effect of the positive PEMs (Figure 4b). Hence, the S/N ratio values took the more sharp turn from the PS plate to the 10-step assembly of PEMs (Figure 4c). The S/N ratio of (PDDA/ PSS)3PDDA was 1.44, which was 11.1 and 3.6 times higher than that of (PDDA/PSS)3 (0.13) and the PS plate (0.4), respectively. Interestingly, the correlation between the S/N ratios on the different substrates was similar to the ratio of OVA to the primary antibody molecules, and the ratio of the (PDDA/ PSS)3PDDA was 11.5 and 2.5 times higher than that of (PDDA/ PSS)3 and the PS plate, respectively. This result strongly suggests that the sensitivity of antigen detection was mainly dependent with the blocking effect per adsorbed primary antibody molecule. In addition, the S/N ratio of the PS plate was between the positive and negative PEMs, considering that the characteristics of the PS plate were intermediate between both PEMs.

The polyelectrolyte multilayers could reach stable over the 6-step assembbvaly due to the surface property was clearly defined the outermost layer of polymer in general. Therefore, we considered that the ELISA system also reaches stable over the 6-step of PEMs. Moreover, in all of the steps of the PDDA/PSS PEMs, the S/N ratio of the (PDDA/PSS)3 and (PDDA/PSS)3PDDA were the smallest and largest, respectively, hence we selected them to estimate the antigen detection. Figure 5 shows the specific signals with the change in antigen concentration from 0.002 to 5 µg/mL. On the (PDDA/PSS)3PDDA substrate, the standard deviations of the signal were small, and moreover had the largest change in signal (the slope of line was the largest) as compared with (PDDA/PSS)3 and the PS plate. Specifically, (PDDA/PSS)3PDDA PEMs bearing a high performance surface could efficiently detect antigen molecules. In contrast, on the (PDDA/PSS)3 PEMs and PS plate, we observed much larger standard deviations, and on some particular occasions, did not obtain any significant differences through the antigen concentration. In Table 3, we compared the slope of the calibration curves and signal change in different concentration range of the antigen solution in Figure 5. Obviously, on the (PDDA/ PSS)3PDDA surface, the calibration curve had the largest slope and signal change, and they are approximately 2 times higher than that of the PS plate which was regardless of low or high concentration range of antigen solution. Taking this result into account, (PDDA/PSS)3PDDA PEMs possess excellent performance for improving the conventional ELISA system due to the efficient blocking effect of nonspecific protein adsorption. On the other hand, the amount of the primary antibody adsorption on the (PDDA/PSS)3PDDA (0.31 µg/cm2) was approximately half of the (PDDA/PSS)3 (0.63 µg/cm2) adsorption. However, the (PDDA/PSS)3PDDA PEMs achieved higher sensitivity and a larger change in the specific signal on antigen detection (Figure 4b). Jiang et al. demonstrated that the desired end-on (Fc fragment closer to surface) orientation was achieved on a positively charged surface, while the head-on and lyingflat orientations were observed on the negatively charged and Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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neutral surfaces, respectively, due to the dipole points from the Fc and (Fab)2 fragments of the antibody.30 Therefore, in this study, we speculated that the primary antibody should be preferentially orientated in an end-on position on the positive PEMs substrate for a favorable response to the antigen, serving to achieve the highest sensitivity of immunoassay. Based on all of the above results, one can deduce the configuration of each protein adsorption on the PS plate, (PDDA/ PSS)3, and (PDDA/PSS)3PDDA, respectively (Figure 6). On the (PDDA/PSS)3 PEMs, even though a large number of primary antibodies were adhered, they did not achieve highly sensitive detection due to the low coverage of the blocking reagent. On the other hand, even though the amount of primary antibody adsorption on the (PDDA/PSS)3PDDA was approximately equal to the PS plate, the coverage of the blocking reagent on the (PDDA/PSS)3PDDA was much greater than that of the PS plate. Therefore, (PDDA/PSS)3PDDA PEMs achieved the highest sensitivity of antigen detection due to its excellent advantages, thus improving the conventional ELISA system such as on the PS plate, which has been the gold standard. CONCLUSIONS Polyelectrolyte multilayers, PDDA/PSS assemblies, were fabricated on PS multiwell plates by a novel alternate drop coating (30) Zhou, J.; Tsao, H. K.; Sheng, Y. J.; Jiang, S. Y. J. Chem. Phys. 2004, 121, 1050–1057.

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process. On the positively charged polyelectrolyte multilayers, we obtained the highest sensitivity of antigen detection due to the efficient inhibition of nonspecific adsorption by the excellent blocking effect, as compared to the conventional PS plate in the ELISA system. We suggest that positively charged polyelectrolyte multilayers could modify a given substrate to easily create various high performance devices in order to improve the conventional ELISA system. ACKNOWLEDGMENT We thank Drs. T. Kida, M. Matsusaki, H. Ajiro, and T. Akagi from Osaka University for their helpful discussions and advice. Part of this study was financially supported by a Grant-in-Aid for the 21st Century COE Program “Center for Integrated Cell and Tissue Regulation (CICET)” from The Ministry of Education, Culture, Sport, Science and Technology, Japan. H.Y.S. was supported by a grant from CICET and a scholarship from the Osaka Foundation of International Exchange. SUPPORTING INFORMATION AVAILABLE Additional information and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 7, 2009. Accepted June 28, 2009. AC900985X