Recognition of Dengue Virus Protein Using Epitope-Mediated

Anal. Chem. 2005, 77, 5140-5143

Recognition of Dengue Virus Protein Using Epitope-Mediated Molecularly Imprinted Film Dar-Fu Tai,*,† Chung-Yin Lin,† Tzong-Zeng Wu,‡ and Li-Kuang Chen§

Department of Chemistry and Institute of Biotechnology, National Dong-Hwa University, Hualien, Taiwan, and Department of Emergency Medicine, Medical School, Buddhist Tzu Chi University, Hualien, Taiwan

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required. Direct multiple clinical sample analysis without pretreatment is the ultimate goal for a protein-detecting array.1-3 The most significant challenge is to separate and detect each protein at the molecular level. Previously, sensor chip construction was based on the proper placement of a natural affinity material able to form strong interactions with the protein, predominantly the antibody,4-7 antigen,8 peptide,9 receptor,10 or aptamer.11 The drawback of using immobilized biomaterials is the inability to obtain them in pure form to load onto the chips2 and control the activity, orientation, reusability, and cost at the same time. * To whom correspondence should be addressed. E-mail: dftai@ mail.ndhu.edu.tw. † Department of Chemistry, National Dong-Hwa University. ‡ Institute of Biotechnology, National Dong-Hwa University. § Buddhist Tzu Chi University. (1) Jenkins, R. E.; Pennington, S. R. Proteomics 2001, 1, 13-29. (2) Kodadek, T. Chem. Biol. 2001, 8, 105-115. (3) Wilson, D. S.; Nock, S. Angew. Chem., Int. Ed. 2003, 42, 494-500. (4) Cooper, M. A.; Dultsev, F. N.; Minson, T.; Ostanin, V. P.; Abell, C.; Klenerman, D. Nat. Biotechnol. 2001, 19, 833-837. (5) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (6) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Nat. Biotechnol. 2001, 19, 866-869. (7) Babacan, S.; Pivarnik, P.; Letcher, S.; Rand, A. G. Biosens. Bioelectron. 2000, 15, 615-621. (8) Robinson, W. H.; DiGennaro, C.; Hueber, W.; Haab, B. B.; Kamachi, M.; Dean, E. J.; Fournel, S.; Fong, D.; Genovese, M. C.; de Vegvar, H. E. N.; Skriner, K.; Hirschberg, D. L.; Morris, R. I.; Muller, S.; Pruijn, G. J.; van Venrooij, W. J.; Smolen, J. S.; Brown, P. O.; Steinman, L.; Utz, P. J. Nat. Med. 2002, 8, 295-301. (9) Zhang, Z.; Zhu, W.; Kodadek, T. Nat. Biotechnol. 2000, 18, 71-74. (10) Lu, H. C.; Chen, H. M.; Lin, Y. S.; Lin, J. W. Biotechnol. Prog. 2000, 16, 116-124. (11) Hermann, T.; Patel, D. J. Science 2000, 287, 820-825.

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Recently, growing interest in mimicking natural receptors using antibody equivalents has increased.12,13 Molecularly imprinted polymers (MIPs) have already been successfully formulated to mimic natural receptors.14,15 Most of the MIPs generated have been used for detecting small compounds. However, attempts to imprint proteins have met with only limited success. These artificial receptors are not as specific as antibodies, but they are far more robust. Template-imprinted nanostructural surfaces16 seem to be a promising way to overcome such difficulties. Epitopemediated imprinting has been reported to recognize small peptides.17 Surface grafting is also an attractive approach.18 However, a combination of these technologies has not yet been reported. Previously, a quartz crystal microbalance (QCM) chip has been successfully used in the detection of proteins.7,10,19 However, its application in the viremia or serological phases are limited by interference. Dengue virus is a newly emerging disease in the world that poses a serious problem in developed and developing countries.20 The clinical symptoms for dengue fever and dengue hemorrhagic fever are atypical.21,22 Effective vaccines or drugs are still unavailable to prevent or cure the diseases caused by the dengue virus.23 It is crucial for a doctor to diagnose dengue fever early with high accuracy, short operation time, and labor-free processing. Dengue viruses belong to BSL 2 (Biosafety Levels) and should be cultured in a P2 laboratory. Dengue viruses were usually UV-irradiated after culture and used for routine serological and PCR assays. Therefore, to develop a novel detection system for identifying dengue virus infection is of great urgency.24, 25 (12) Kodadek, T.; Reddy, M. M.; Olivos, H. J.; Bachhawat-Sikder, K.; Alluri, P. G. Acc. Chem. Res. 2004, 37, 711-718. (13) Srinivasan, N.; Kilburn, J. D. Curr. Opin. Chem. Biol. 2004, 8, 305-310. (14) Snowden, T. S.; Anslyn, E. V. Curr. Opin. Chem. Biol. 1999, 3, 740-746. (15) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (16) Shi, H.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (17) Rachkov, A.; Minoura, N. J. Chromatogr., A 2000, 889, 111-118. (18) Bossi, A.; Piletsky, S. A.; Piletska, E. V.; Righetti, P. G.; Turner, A. P. F. Anal. Chem. 2001, 73, 5281-5286. (19) Su, C. C.; Wu, T. Z.; Chen, L. C.;Yang, H. H.; Tai, D. F. Anal. Chim. Acta 2002, 479, 117-123. (20) Monath, T. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2395-2400. (21) Halstead, S. B.; O’Rourke, E. J. Nature 1977, 265, 739-741. (22) Gubler, D. J. Clin. Microbiol. Rev. 1998, 11, 480-496. (23) Chang, G.-J. J.; Davis, B. S.; Hunt, A. R.; Holmes, D. A.; Kuno, G. Ann. N. Y. Acad. Sci. 2001, 951, 272-285. (24) Vaughn, D. W.; Nisalak, A.; Solomon, T.; Kalayanarooj, S.; Dung, N. M.; Kneen, R.; Cuzzubbo, A.; Devine, P. Am. J. Trop. Med. Hyg. 1999, 60, 693698. (25) Teles, F. R. R.; Prazeres, D. M. F.; Lima-Filho, J. L. Rev. Med. Virol. In press. 10.1021/ac0504060 CCC: $30.25

© 2005 American Chemical Society Published on Web 07/07/2005

Scheme 1. Comparison of Epitope-Mediated Imprinting with Protein-Engaged Imprinting

Recently, we have demonstrated the detection of peptides using MIP-QCM.26 Herein, we describe the epitope approach to synthesize molecular imprinting film onto a sensor chip. As shown in Scheme 1, the epitope-mediated imprinting process is compared with protein-engaged imprinting on the surface. Our approach makes it possible to obtain an imprinted polymer selective to a specific protein. This new method offers several advantages over traditional MIP methods. First, embedding and bleeding the target protein are avoided. Second, the nonspecific interaction between the MIP and target protein is minimized. Third, all of the proteins bound to the MIP are arranged in proper orientation. Fourth, analyte washout is quicker and more efficient to allow maximum reusability. EXPERIMENTAL SECTION (Boc-L-Cys)2, acrylic acid, acrylamide, pepsin, IgG from rat serum, and bovine albumin were obtained from Sigma-Aldrich (St. Louis, MO). N-Benzylacrylamide was purchased from Lancaster (26) Lin, C.-Y.; Tai, D.-F.; Wu, T.-Z. Chem. Eur. J. 2003, 9, 5107-5110.

(Lancashire, U.K.). The 15-mer peptide27 derived from nonstructural protein 1 (NS1) of a Japanese encephalitis virus was synthesized by a peptide synthesizer. (N-Acr-L-Cys-NHBn)2 was synthesized from (Boc-L-Cys)2.26 The buffer used for all experiments was a PBS (20 mM NaH2PO4, pH 4.0). The QCM was obtained from Tai-Tien Electronic Co. (Taipei, Taiwan) with a reproducibility of (1 Hz. The QCM consisted of an 8-mm-diameter disk made from an AT-cut 9-MHz quartz crystal with gold electrodes on both sides (diameter, 4.5 mm; area, 15.9 mm2) of the crystal. Preparation of Imprinted Polymer-Coated QCM. The QCM disks were immersed in a 10 µM solution of (N-Acr-L-CysNHBn)2 in HPLC-grade acetonitrile for 16 h and then rinsed exhaustively with acetonitrile. A solution of acrylic acid (55 µmol), acrylamide (55 µmol), N-benzylacrylamide (110 µmol), and 15mer peptide (3 µmol) were mixed in 0.3 mL of solution (acetonitrile/20 mM, pH 4.0 phosphate buffer ) 1:1). After depositing 4 µL of the aliquot on top of the (N-Acr-L-Cys-NHBn)-gold electrode, the chip was placed horizontally into a 20-mL vial containing acetonitrile (3 mL). The vial was screwed tightly and irradiated with UV light at 350 nm for 6 h. The polymer, which Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

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was formed as a thin film on the gold surface, was washed with 20 mM phosphate buffer (pH 3-4) to remove the template. This was followed by a wash with methanol and drying. The thickness of the films was measured using a surface profiler from Veeco Inc. (Dekatak3 ST). Biosensor System. The flow injection system contained a HPLC pump (model L7110, Hitachi, flow rate 0.1 mL min-1), home-built flow cell, sample injection valve (model 1106, OMNIFIT), home-built oscillation circuit (including oscillator and frequency counter), and a personal computer. The polymer-coated QCM was fixed between two O-rings and inserted into the flow cell. Only one side of the QCM was in contact with the liquid. Sodium phosphate buffer (20 mM, pH 4.0) was used for circulating, washing, and testing. To equilibrate the newly imprinted chips quickly, 100-µL solutions, including alkaline (pH 9 PBS), neutral (distilled water), and acidic (5% acetic acid in distilled water), were injected into the flow cell during circulation. RESULTS AND DISCUSSION The NS1 protein is found associated with membranes in the cytoplasm of infected cells. When cells are infected with the dengue virus, they display NS1 on the cell surface or secrete NS1 into the blood. Hence, many NS1 proteins exist in the blood specimen during the viremia phase.28 In addition, the anti-NS1 antibodies possess the ability to protect animals against a virus challenge. A 15-mer peptide containing 90-95 of the Japanese encephalitis virus nonstructural protein 1 Thr-Glu-Leu-Arg-TyrSer-Trp-Lys-Thr-Trp-Gly-Lys-Ala-Lys-Met was chosen as the template. This peptide was reported as the linear epitope of the NS1 protein.27 The epitope of antibody D2/8-1 was mapped to this 15-mer peptide, in which consensus linear sequence [WK(A/T)WGK] is present in the NS1 of the Japanese encephalitis and dengue virus. On the basis of previous experiments,26 N,N′-diBoc-L-cystine dibenzylamide was self-assembled onto the gold surface of a QCM chip. A monomer solution, containing acrylic acid/acrylamide/ N-benzylacrylamide and the pentadecapeptide was added onto the QCM chip and irradiated with UV to form polymeric thin films with an average thickness of 70 nm. It is not necessary to add an initiator for UV-irradiated photopolymerization. Although we do not know the exact mechanism, a thiol-ene photopolymerization29 is suspected. The formatted chips were treated with alkaline, neutral, and acidic solutions to wash out the template. The MIP-grafted 15-mer peptide chips were then tested for their ability to rebind templates (1613 Mw) and their mother protein (24 000 Mw). It has been known28 that the mature form of the NS1 protein is a dimer (48 000 Mw). Oligomerization of the NS1 protein is a general feature of flavivirus infection. The dimer is very stable at pH 6 but gradually dissociates to a monomer at lower pH. A considerable amount of dimmer will be converted to monomer at pH 3. However, most of the protein remains as a dimer at pH 4. The chip was inserted into a flow cell circulating (0.5 mL/min) with 20 mM sodium phosphate buffer, pH 4.0. The (27) Chen, L. C.; Liao, C. L.; Lin, C. G.; Lai, S. C.; Liu, C. I.; Ma, S. H.; Huang, Y. Y.; Lin, Y. L. Virology 1996, 217, 220-229. (28) Winkler, G.; Randolph, V. B.; Cleaves, G. R.; Ryan, T. E.; Stollar, V. Virology 1988, 162, 187-196. (29) Cramer, N. B.; Scott, J. P.; Bowman, C. N. Macromolecules 2002, 35, 53615365.

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Figure 1. Analyte concentration effect on the MIP-QCM chip frequency shifts (NIP, nonimprinted polymer).

readsorption of the 15-mer peptide chips were evaluated by injecting 100 µL of the pentadecapeptide solutions at different concentrations, respectively. We also tested the culture supernatant of the NS1 protein solution and the purified NS1 protein solution at various concentrations. The data obtained were plotted with the saturation equation for specific binding (B ) Bmaxc/(Kd + c), B ) H/Mw, where c is the concentration of protein, B is the fraction of sites bound, Mw is the molecular weight of the analyte, and H is the frequency shifts in the QCM). Figure 1 shows the affinities of the pentadecapeptide and NS1 protein to MIPs as a function of the analyte concentration used. From the binding experiments, the polymers imprinted with a peptide efficiently recognized both the template and proteins that possessed the same epitope part of the structure. It was demonstrated that although other parts of the impurity might influence the molecular recognition process, the frequency shift was correlated for purified and unpurified protein solutions. To remove the NS1 protein from the QCM chip in a more efficient way, the polymers were washed with 5% acetic acid/0.5% Tween-20/25 mM urea as part of the activation/regeneration procedure.18 It was evident that the size of the molecule and its charge affected the imprinting efficiency. The dissociation constants for the MIP to the 15-mer peptide and NS1 protein were measured using the modified equation, Hmax/H ) Kd/c + 1. For the pentadecapeptide, the dissociation constant was Kd ) 0.6 nM, which was lower than that for the nonapeptide (oxytocin and vasopressin).26 The frequency shifts observed for the pentadecapeptide were also higher than that for the protein solutions. As shown in Figure 2, purified and unpurified dengue NS1 proteins exhibited much lower Kds, 0.04 and 0.09 nM, respectively. All Kds were in the nanomolar range, indicating a strong polymertemplate interaction resulting from the multipoint attachment of the NS1 protein to the 15-mer peptide chip. The data were also compared with the Kd (0.05 nM) of the purified NS1 protein to the chip immobilized with monoclonal antibodies.19 The effective amount of antibody decreases upon immobilization, while selfassembly MIPs took advantage of the chip surface direct coating and performed slightly better in this experiment. Although it is still unclear which one had the higher affinity in free form, their Kd values must be comparable. The experimental setup to confirm the target protein in an unknown mixture is schematically shown in Scheme 1. We found monoclonal antibody D2/8-1 27 serves this purpose well to bind the unbounded epitope site of the NS1 protein dimer on the chip.

Table 1. Frequency Shifts Observed upon Ternary Complex Formation 1st injection of unpurified NS1 protein solution

2nd injection of antibody (1 µg/mL)a

concn of NS1 protein (ng/mL)a

frequency shift (Hz)

frequency shift (Hz)

50 000 5000 500 50 5 0.5

67 40 26 12 5 0

15 14 10 0 0 0

a

100 µL per injection.

Figure 2. Comparison of the binding effects of NS1 protein to MIP and antibody.19

was further investigated. The MIP-NS1 protein complex was formed in the presence of various amounts of the NS1 protein. The frequency shift values are shown in Table 1. The results indicate that the chip’s ability to bind the antibody increases steeply for the attached NS1 protein at higher concentrations, while it rests unchanged for MIPs prepared in the presence of an NS1 protein concentration below 50 ng/mL. This situation could be partially explained by the steric repulsion of the antibody to the MIP surface upon complexion with the NS1 protein. The MIP-grafted 15-mer peptide chips were also tested for their ability to bind with other proteins. Injection of a solution of pepsin solution (5 ng/mL), IgG from rat serum (100 ng/mL), or bovine albumin (5 µg/mL) was unable to induce frequency shifts on the QCM.

Figure 3. Consecutive binding of the NS1 protein and NS1 antibody to the MIP chip.

Both 15-mer and antibody D2/8-1 recognize the NS1 protein at the same region. The ternary complex (sandwich) thus appears to play a critical role in protein recognition using the epitopetemplated polymer in contrast to the highly cross-linked MIPs synthesized using the conventional approach. The frequency response of the antibody on MIP-NS1 protein complex binding is shown in Figure 3. At first, 100 µL of the virus antigen dilutions27 was injected into the center of the QCM disk. After 15 min following the first injection, 100 µL of purified monoclonal antibody D2/8-1 (1 µg/mL) was also injected. The affinity of the monoclonal antibody to the chip surface was detected by QCM. Their ability to adsorb the NS1 protein on the chip at different concentrations was measured. The possible role of the free-standing MIP to bind the antibody was also tested. It was found that the affinity of the antibody to the MIP without the NS1 protein was not detectable (Table 1). These results indicate that the synthetic materials have selective recognition and reveal the absolute interactions between the MIPs, protein and antibody. The influence of the NS1 protein concentration on the ability to form MIPs/NS1 protein dimer/monoclonal antibody sandwich

CONCLUSION The efficiency of the epitope-templating procedure for generating surfaces with high affinity toward template molecules and target proteins was demonstrated. The presented surface-imprinting and epitope-mediated technique modification using a masssensitive transducer is an ideal in situ analytical system for studying the protein-protein interactions. It provides a convenient in vitro cellular assay for quantitatively recognizing proteins. This assay offers a number of advantages (specific binding, small samples required, no separation and purification procedures). Early serological detection of dengue and other viral proteins can be performed directly. This method could also serve as a tool to detect the existence of antibodies. ACKNOWLEDGMENT We thank the Taiwan National Institute of Preventive Medicine for providing DEN-2 virus and serum. The Taiwan National Science Council supported this work (NSC 91-2323-B-259-002).

Received for review March 8, 2005. Accepted June 13, 2005. AC0504060

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