Anal. Chem. 2005, 77, 4282-4285
Correspondence
SPR Sensor Chip for Detection of Small Molecules Using Molecularly Imprinted Polymer with Embedded Gold Nanoparticles Jun Matsui,†,‡ Kensuke Akamatsu,†,‡ Noriaki Hara,‡ Daisuke Miyoshi,† Hidemi Nawafune,†,‡ Katsuyuki Tamaki,‡ and Naoki Sugimoto*,†,‡
Frontier Institute for Biomolecular Engineering Research (FIBER) and Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan
Molecularly imprinted polymer gel with embedded gold nanoparticle was prepared on a gold substrate of a chip for a surface plasmon resonance (SPR) sensor for fabricating an SPR sensor sensitive to a low molecular weight analyte. The sensing is based on swelling of the imprinted polymer gel that is triggered by an analyte binding event within the polymer gel. The swelling causes greater distance between the gold nanoparticles and substrate, shifting a dip of an SPR curve to a higher SPR angle. The polymer synthesis was conducted by radical polymerization of a mixture of acrylic acid, N-isopropylacrylamide, N,N′-methylenebisacrylamide, and gold nanoparticles in the presence of dopamine as model template species on a sensor chip coated with allyl mercaptan. The modified sensor chip showed an increasing SPR angle in response to dopamine concentration, which agrees with the expected sensing mechanism. Furthermore, the gold nanoparticles were shown to be effective for enhancing the signal intensity (the change of SPR angle) by comparison with a sensor chip immobilizing no gold nanoparticles. The analyte binding process and the consequent swelling appeared to be reversible, allowing one the repeated use of the presented sensor chip. Since a commercially available surface plasmon resonance (SPR) sensor has been introduced, a number of sensor systems have been developed and have found practical applications in various chemical fields such as biochemistry and analytical chemistry.1 The sensors detect the change in dielectric constants near noble metal film on the sensor chips; therefore, most of the sensor design is concerned with surface modification, e.g., immobilization of molecular recognition elements selective to an * To whom correspondence should be addressed. E-mail: sugimoto@ konan-u.ac.jp. † Frontier Institute for Biomolecular Engineering Research (FIBER). ‡ Department of Chemistry, Faculty of Science and Engineering. (1) (a) Homola, J.; Yee, S. S.; Gauglitz, G. Sens. Actuators 1999, B54, 3-15. (b) Silin, V. Plant, A. Trends Biotechnol. 1997, 15, 353-359.
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analyte in question.2 To date, numerous recognition systems have been devised employing DNA, proteins, other natural polymers, and synthetic polymers. Only a few tactics, however, have been addressed in improving the sensitivity although conventional SPR sensors are not sensitive for small-molecule detection. Pitner et al. utilized conformational changes of macromolecules, such as maltose binding protein and tissue transglutaminase, on analyte binding.3 Another literarily reported strategy employed probe molecules with high molecular weights or high refractive indexes, which comes in proximity to the sensor chip surface triggered by analyte binding.4 For example, Keating et al. reported a more than 1000-fold enhancement in sensitivity using Au nanoparticleDNA conjugate as a probe molecule that binds an analyte DNA molecule captured on a sensor chip.5 These examples show that a key to highly sensitive detection is development of a signal transducing system converting the molecular recognition to another event accompanied by larger changes in dielectric constants. It was therefore envisioned that the cooperation of the above-mentioned two strategies, using both an analyte binding macromolecule and an Au nanoparticle, could afford a sensor chip for sensitive small-molecule detection without requiring probe reagents.6 We have recently reported a polymer gel with immobilized Au nanoparticle as colorimetric sensing material,7 which was prepared by a molecular imprinting technique.8,9 The molecularly (2) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag: Berlin, 1988; Vol 111. (3) (a) Gestwicki, J. E.; Hsieh, H. V.; Pitner, J. B. Anal. Chem. 2001, 73, 57325737. (b) Sota, H.; Hasegawa, Y.; Iwakura, M. Anal. Chem. 1998, 70, 20192024. (4) Karlsson, R. Anal. Biochem. 1994, 221, 142-151. (5) (a) Hutter, E.; Pileni, M.-P. J. Phys. Chem. B 2003, 107, 6497-6499. (b) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775138. (6) Very recently, immobilization on a sensor chip (gold nanoislands) of pHresponsive polymer brushes attached with Au nanoparticles has been reported for construction of a transmission surface plasmon resonance (TSPR) sensor. This study proves the validity of the concept of our study to use both macromolecule and Au nanoparticle for fabricating an SPR sensor chip. Tokareva, I.; Minko, S.; Fendler, J. H.; Hutter, E. J. Am. Chem. Soc. 2004, 126, 15950-15951. (7) Matsui, J.; Akamatsu, K.; Nishiguchi, S.; Miyoshi, D.; Nawafune, H.; Tamaki, K.; Sugimoto, N. Anal. Chem. 2004, 76, 1310-1315. 10.1021/ac050227i CCC: $30.25
© 2005 American Chemical Society Published on Web 05/25/2005
Figure 1. Schematic representation of Au-MIP/MIP-coated SPR sensor chip for detection of an analyte, dopamine.
imprinted polymer with immobilized Au nanoparticle (Au-MIP) exhibited selective binding of a small molecule accompanied by swelling, which caused the greater distance between the immobilized Au nanoparticles. The varied proximity of the nanoparticles was spectroscopically read out.7 The result encouraged us to apply Au-MIP to SPR sensors as a recognition and signaling element based on a scheme shown in Figure 1. MIP swells by incorporating water in accordance with analyte binding, which would lead to significant changes in dielectric constant near the Au substrate surface. More importantly, the swelling would cause increasing distance between the Au nanoparticle within the polymer gel and Au film on the sensor chip surface, which would result in drastic enhancement in the degree of SPR angle shift. On this basis, we developed here an Au-MIP modified SPR sensor chip for small-molecule detection and examined for sensing dopamine as a proof-of-principle analyte. EXPERIMENTAL SECTION Chemicals and Instrument. Acrylic acid (AA), 2,2′-azobis(2,4-dimethlvarelonitrile), dopamine, N-isopropylacrylamide (NIPA), N,N′-methylenbisacrylamide (BIS), tetraoctylammonium bromide (TOAB), and hydrogen tetrachloroaurate(III) (HAuCl4‚4H2O) were purchased from Wako Pure Chemicals (Osaka, Japan). Allyl mercaptan, decanethiol, 11-mercaptoundecanoic acid (MUA), sodium borohydride were obtained from Sigma Aldrich Japan (Tokyo, Japan). Gold sputtered cover glass (13 × 20 mm) as a sensor chip was purchased from Nippon Laser & Electronics Lab (Nagoya, Japan). Surface plasmon resonance measurements were conducted using SPR 670 (Nippon Laser & Electronics Lab). (8) (a) Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H., Eds. Molecular Imprinting-From Fundamentals to Applications; Wilely-VCH: Weinheim, Germany, 2002. (b) Sellergren, B. Molecularly Imprinted Polymers: ManMade Mimics of Antibodies and their Application in Analytical Chemistry; Elsevier: Amsterdam, 2001. (c) Bartsch, R. A.; Maeda, M. Molecular and Ionic Recognition with Imprinted Polymers; ACS Symposium Series 703; The American Chemical Society: Washington, DC, 1998. (d) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (e) Haupt, K.; Mosbach, K. Trends Biotechnol. 1998, 16, 468-475. (f) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (g) Shea K. J. Trends Polym. Sci. 1994, 2, 166. (9) Watanabe, M.; Akahoshi, T.; Tabata, Y.; Nakayama, D. J. Am. Chem. Soc. 1998, 120, 5577-5578.
Preparation of Size-Controlled Au Nanoparticles. The nanoparticle was prepared by a previously reported procedure.10 An aqueous solution of HAuCl4‚4H2O (15 mM, 200 mL) and TOAB (6.0 mmol) in toluene (400 mL) were mixed. Into the mixture, MUA (3.0 mmol) in toluene (100 mL) was gradually added with vigorous stirring. An aqueous solution of NaBH4 (0.30 M, 100 mL) was then added dropwise. After the mixture was stirred for 1 h, the organic phase was separated and washed with distilled water. After removal of the solvent, the resultant black solid was heattreated at 155 °C at the heating rate of 2 °C min-1 for 30 min. The heat-treated product was dissolved in 50 mL of toluene, mixed with 800 mL of chloroform to remove excess TOAB and MUA, and then filtered off to give the Au nanoparticles. Preparation of Au-MIP-Coated Sensor Chips. Sensor chips were immersed in allyl mercaptan (10 mM, ethanol) or decanethiol (10 mM, ethanol) overnight. Between the allyl mercaptan-modified chip and the decanethiol-modified one, a prepolymerization mixture was heated (60 °C, 4 h), which consists of dopamine (60 µmol), AA (233 µmol), NIPA (943 µmol), BIS (58 µmol), 2,2′azobis(2,4-dimethlvarelonitrile) (0.2 mg), and DMSO (1 mL). Putting apart from the decanethiol-modified chip, a MIP-coated chip was obtained. A prepolymerization mixture with Au nanoparticle (29 mg) was further immobilized on the MIP-coated sensor chip to yield an Au-MIP/MIP-coated sensor chip 1. Sensor chips were also prepared by modification with Au-MIP/MIP of lower Au density (40% to 1) (2), with MIP/MIP (3), and with 11-mercaptoundecanoic acid alone (4) that bears a carboxyl group. RESULTS AND DISCUSSION A dopamine-imprinted polymer-coated sensor chip 1 was constructed by radical polymerization on an allyl mercaptanmodified gold substrate. Modification was conducted in two steps; the gold substrate was first modified with a MIP layer (without Au nanoparticle) and then with Au-MIP (with Au nanoparticle). The two-step modification was conducted to avoid immobilization of Au nanoparticles in too close proximity to the Au film surface because such Au nanoparticles may cause poor sensitivity. A scanning electron microscope (SEM) image of 1 confirmed the immobilization of the polymer layer and indicated that the thickness of the polymer layer (Au-MIP + MIP) is ∼6 µm (in a vacuum) (Figure 2). Prior to dopamine analysis, the SPR angle was measured on the four sensor chips 1-4 (see Experimental Section). Typical SPR curves obtained in water at 30 °C are shown in Figure 3. Comparing 3 and 4, it was suggested that modification with MIP was effective for increasing the SPR angle. The results are consistent with a previously reported acrylamide-coated SPR sensor chip; the coating was detected by an increase of the SPR angle.11 Sensor chips 1 and 2 showed further increase in the SPR angle. The result could be due to coupling between the localized surface plasmon of the Au nanoparticle and surface plasmon polarization,2 according to a literature reported phenomenon that the increased proximity of the Au nanoparticle to the Au substrate exhibits an increase in SPR angle.5 In addition, 1 exhibited a higher SPR angle than 2, possessing a lower density of Au (10) Akamatsu, K.; Hasegawa, J.; Nawafune, H.; Katayama, H.; Ozawa, F. J. Mater. Chem. 2002, 12, 2862-2865. (11) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 8196-8202.
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Figure 4. (a) A typical sensorgram with Au-MIP/MIP immobilized SPR sensor chip (1): sample, dopamine hydrochloride (20 µL); a flow rate, 7 µL min-1; temperature, 30 °C. Dashed lines indicate the period when the sample contacted the sensor chip. (b) A calibration curve to convert the SPR angle shift to the dopamine concentration.
Figure 2. SEM image of a sensor chip 1: (a) side view, (b, c) slant views. Thickness of the polymer layer was determined to be ∼6 µm. No clear boundary between Au-MIP and MIP was observed, suggesting that polymerization of the second layer (Au-MIP) took place, to some degree, in the matrix of the first layer (MIP). Partial detachment of the polymer layer observed in the slant views (b, c) occurred when the sensor chip was cut into halves for SEM measurement.
Figure 3. Typical SPR curves of the surfaces modified with AuMIP/MIP gel (1), with Au-MIP/MIP of lower density nanoparticle (2), with MIP/MIP (3), and with 11-mercaptoundecanoic acid (4) in water at 30 °C. A commercially available SPR sensor (Nippon Laser & Electronics Lab., SPR-670) was used for all the measurements.
nanoparticles, supporting the consideration that the origin of the higher SPR angle is the incorporation of the Au nanoparticle into the MIP matrix. Polymer coating with an Au nanoparticle density higher than that of 1 was examined; however, the resultant SPR angle was out of the effective range of the employed SPR sensor (>79°). An SPR angle was also measured at 20 °C to investigate temperature-dependent behaviors of the sensor chips because AuMIP is known to exhibit swelling in response to the low temperature.7 Sensor chip 3 exhibited a dip at 72.9° at 20 °C, 4284 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005
showing a lessened SPR angle by -0.87° compared to that at 30 °C. Furthermore, 1 exhibited a significantly larger difference (-2.48°) at a 10 °C decrease. The response was in contrast to when the bare (no MIP coat) sensor chip 4 was examined; 4 exhibited a dip at 70.1° at 20 °C, showing a 0.16° increase in the SPR angle, confirming that the observed response was attributed to swelling of MIP. The result that the swelling of MIP could be monitored by the SPR angle suggests that detection of dopamine is expected to be possible with this system because the analyte induces swelling of MIP as well as decreased temperature. Also, the larger SPR angle shift with 1 suggests that sensitivity enhancement can be expected due to the Au nanoparticle shifting away from the chip surface. The response of sensor chips 1-4 was measured upon injection of 10 µM dopamine. While sensor chip 4 exhibited no significant response, 3 exhibited a decrease of 0.185° in the SPR angle. Furthermore, sensor chips 1 and 2 showed a greater shift in the SPR angle, -0.363° and -0.477°, respectively, showing the effectiveness of the Au nanoparticle for enhancing the sensitivity. The direction of the observed shift was consistent with that observed by decreasing temperature, which suggests that the origin of the response is swelling of MIP and Au-MIP. The results of sensor chips 1 and 2 again support that the incorporation of Au nanoparticles is useful for enhancing the sensitivity of the MIPcoated sensor chip. The sensitivity of 1 was examined upon injection of various concentrations of dopamine, ranging from 1 nM to 1 mM. As shown in a typical sensorgram upon injection of 10 µM, 100 µM, and 1 mM (Figure 4a), the responses depend on the sample concentration. The sensorgram also suggests that both the analyte binding process and the consequent swelling are reversible, allowing one the repeated use of the presented sensor chip. A calibration was prepared for assessing the sensitivity as shown in Figure 4b. It would be noted that even a nanomolar sample exhibited a significant shift of SPR angle. The results suggest that cooperative use of an analyte binding macromolecule and an Au nanoparticle was effective for development of sensitive sensor chips because MUA-modified sensor chip 4 exhibited only a 0.05° shift at 10 mM dopamine. The sensitivity of the Au-MIP-based SPR sensor system can be also emphasized by comparison with the previous colorimetric system using a spectrophotometer, in which Au-MIP exhibited significant spectroscopic changes at 5 µM or higher concentrations.7 In this preliminary study, optimiza-
tion of the conditions of the polymer synthesis has not been conducted. Because performance of the sensor depends on molecular recognition ability and swelling capacity of Au-MIP, it would be necessary to consider both for increasing the sensitivity by altering the conditions of the polymer synthesis. CONCLUSION We have shown the response of the Au-MIP/MIP-modified SPR sensor chip to an analyte, dopamine, demonstrating the potential usefulness of Au-MIP as SPR sensing material for sensitive detection of low molecular weight species. Furthermore, it is promising that higher concentrations of Au nanoparticle will further enhance the sensitivity. Unfortunately, such attempts were not made in this study because the SPR angle was too high to be measured with the employed SPR sensor system. Instrumental
design to utilize Au-MIP with higher Au density for composing a highly sensitive system is underway. ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research and Academic Frontier Project for Private Universities from MEXT (Ministry of Education, Culture, Sports, Science and Technology), Japan.
Received for review February 5, 2005. Accepted April 27, 2005. AC050227I
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