Sensing Capabilities of Colloidal Gold Modified with a Self-Assembled

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Sensing Capabilities of Colloidal Gold Modified with a Self-Assembled Monolayer of a Glucose-Carrying Polymer Chain on a Glass Substrate† Shinta Morokoshi, Kazuhiko Ohhori, Kazuya Mizukami, and Hiromi Kitano* Department of Chemical and Biochemical Engineering, Toyama University, Toyama, 930-8555 Japan Received March 26, 2004. In Final Form: June 18, 2004 A disulfide-carrying polymer with pendent glucose residues (poly(2-methacryloyloxyethyl D-glucopyranoside)) was obtained by using a benzyl N,N-diethyldithiocarbamoyl derivative which shows the abilities of initiation, chain transfer, and termination (iniferter). The disulfide-carrying polymer was accumulated on a colloidal Au-immobilized glass substrate, and the usefulness of the polymer as a sensing element of concanavalin A (Con A) was examined by using a UV-visible spectrophotometer with the help of surface plasmon resonance. The sensor showed a concentration-dependent specific binding of Con A with a detection limit of 1.9 nM, and furthermore, it had a very high stability at high ionic strength. The polymer-coated device examined here was not only useful as a simple biosensor chip but is also expected to expand our knowledge of interfacial phenomena by introducing various functional polymers on colloidal Au.

Introduction Surface plasmon resonance (SPR) is a collective electron oscillation that occurs in thin metal layers which can only be excited by evanescent waves. The propagation constant ksp of such a wave depends on both the permittivity and thickness of the metal and that of any dielectric layer on top of it. Currently, there is a great interest in the optical properties of noble metal colloidal particles such as Au or Ag. This is partly due to their usefulness as functional materials in applications including, but not limited to, optical devices,1,2 surface-enhanced spectroscopies,3-11 and chemical and biological sensors.12-16 Previous studies showed that Au colloids can be selfassembled from solution onto a functionalized glass surface * To whom correspondence should be addressed. E-mail: kitano@ eng.toyama-u.ac.jp. † Presented at the Regional Meeting of the Society of Polymer Science, Japan, at Kanazawa University in October 2003. (1) Dirinx, Y.; Bastiaansen, C.; Caseri, W.; Smith, P. Adv. Mater. 1999, 11, 223. (2) Kroschwitz, J. I.; Howe-Grant, M. Glass, 4th ed.; Kroschwitz, J. I., Howe-Grant, M., Eds.; John Wiley & Sons: New York, 1994; Vol. 12, p 569. (3) Jensen, T. R.; van Duyne, R. P.; Johnson, S. A.; Maroni, V. A. Appl. Spectrosc. 2000, 5, 371. (4) Wadayama, T.; Suzuki, O.; Takeuchi, K.; Seki, H.; Tanabe, T.; Suzuki, Y.; Hatta, A. Appl. Phys. A 1999, 69, 77. (5) Tarchya, P. J.; DeSaja-Gonzalez, J.; Rodriguez-Llorente, S.; Aroca, R. Appl. Spectrosc. 1999, 53, 43. (6) Emory, S. R.; Nie, S. J. Phys. Chem. B 1998, 102, 493. (7) Yang, W. H.; Hulteen, J. C.; Schatz, G. C.; van Duyne, R. P. J. Chem. Phys. 1996, 104, 4313. (8) Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.; Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.; Natan, M. J. Science 1995, 267, 1629. (9) Pipino, A. C. R.; Schatz, G. C.; van Duyne, R. P. Phys. Rev. B 1996, 53, 4162. (10) van Duyne, R. P.; Hulteen, J. C.; Treichel, D. A. J. Chem. Phys. 1993, 99, 2101. (11) Meriaudeau, F.; Downey, T. R.; Passian, A.; Wig, A.; Ferrell, T. L. Appl. Opt. 1998, 37, 8030. (12) Okamoto, T.; Yamaguchi, I.; Kobayashi, T. Opt. Lett. 2000, 25, 372. (13) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504. (14) Cheng, S.-F.; Chau, L.-K. Anal. Chem. 2003, 75, 16. (15) Haes, A. J.; van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 10596. (16) Takei, H.; Himmelhaus, M.; Okamoto, T. Opt. Lett. 2002, 27, 342.

to give a monolayer,8,17,18 where the assembly is stabilized by strong attractive colloid-surface interactions and laterally by repulsive colloid-colloid electrostatic interactions. More recently, Okamoto et al.12 showed that the absorbance of an immobilized monolayer of gold colloids is sensitive to the refractive index of the surrounding medium. Moreover, Nath and Chilkoti13 showed that a colloidal Au monolayer can be prepared on glass by selfassembly from solution, and the immobilized Au particles can be subsequently functionalized with biotin. They further demonstrated a new label-free optical sensor that can observe biomolecular interactions in real time at the surface. In this research, we have prepared optical biosensors after the method of Nath and Chilkoti.13 Additionally, we constructed the sensor chip using the iniferter method. “Iniferter”, which has three functions, initiation, chain transfer, and termination, has been often used in living radical polymerization owing to its simplicity and easy of use.19-24 For example, N,N-diethyldithiocarbamoyl (BDC) derivatives have been extensively studied as iniferters.19-22,24 Previously, we have synthesized a novel phospholipid carrying a BDC group as the iniferter and prepared novel block telomer-carrying phospholipids by the successive photoirradiation of various vinyl monomers in the presence of the iniferter.25 Alkyl or aromatic disulfides and thiols form close-packed and well-ordered monolayers on gold or silver surfaces, (17) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735. (18) Grabar, K. C.; Brown, K. R.; Keating, C. D.; Stranick, S. J.; Tang, S. L.; Natan, M. J. Anal. Chem. 1997, 69, 471. (19) Otsu, T.; Yoshida, M. Makromol. Chem., Rapid Commun. 1982, 3, 127, 133. (20) Otsu, T.; Yamashita, K.; Tsuda, K. Macromolecules 1986, 19, 287. (21) Otsu, T.; Matsunaga, T.; Kuriyama, A.; Yoshioka, M. Eur. Polym. J. 1989, 25, 643. (22) Doi, T.; Matsumoto, A.; Otsu, T. J. Polym. Sci., Polym. Chem. Ed. 1994, 32, 2911. (23) Okawara, M.; Nakai, T.; Morishita, K.; Imoto, E. Kogyo Kagaku Zasshi 1964, 67, 2108. (24) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622. (25) Kitano, H.; Chibashi, M.; Nakamata, S.; Ide, M. Langmuir 1999, 15, 2709.

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Scheme 1.

Morokoshi et al.

Preparation of Polymer with Many Pendent Glucose Residues Using the Disulfide-Carrying Iniferter (Cys-BDC)

so-called self-assembled monolayers (SAMs).26-31 Because of their structural analogy to biomembranes,35-37 ease of preparation, and apparent stability,35-44 SAMs of dialkyl disulfides and alkanethiols have been widely used as cell mimetic membranes in recent years. ω-Mercaptoalkanoic acid45 and ω-mercapto-polymer chains with many pendent glucose residues,46 for example, were introduced onto silver colloids as SAMs to investigate the interaction between biomembranes and proteins. In addition, SAMs of cyclodextrin derivatives were constructed on a gold electrode as sensing devices by taking advantage of the ability of cyclodextrin in molecular recognition.47-52 A SAM of a specific substrate for R-chymotrypsin was also constructed on a gold electrode for the detection of the enzyme.53

Recently, we prepared a sugar and carboxylic acid carrying block telomer using a disulfide-carrying iniferter (Cys-BDC, Scheme 1), and a SAM of the block telomer was constructed on Au or Ag surfaces.54 The block telomer modified metal surface showed a response to external stimuli (pH and lectin). We report here that the iniferter Cys-BDC can be used to construct SAMs of functional polymers on colloidal Auimmobilized glass substrates. The prepared polymercoated device was not only useful as a very simple and highly sensitive biosensor chip but also can be expected to expand our knowledge of interfacial phenomenon using various functional polymers on colloidal Au. Experimental Section

(26) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (27) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358. (28) Proter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (29) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (30) Bain, C. D.; Whitesides, G. M. Adv. Mater. 1989, 1, 506. (31) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682. (32) Garrett, R. H.; Grisham, C. M. In Biochemistry; Saunders College Publishing: Fort Worth, 1995. (33) Gennis, R. B. In Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989. (34) Voet, D.; Voet, J. G.; Pratt, C. W. In Fundamentals of Biochemistry; John Wiley & Sons: New York, 1999. (35) Spinke, J.; Lileym, M.; Gunder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821. (36) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (37) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877. (38) Scho¨nher, H.; Vancso, G. J.; Huisman, B.-H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Langmuir 1997, 13, 1567. (39) Schierbaum, K.-D.; Weiss, T.; van Velzen, E. U. T.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go¨pel, W. Science 1994, 265, 1413. (40) Huisman, B.-H.; Kooyman, R. P. H.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Adv. Mater. 1996, 8, 561. (41) Jeon, N. L.; Finnie, K.; Branshaw, K.; Nuzzo, R. G. Langmuir 1997, 13, 3382. (42) Flink, S.; Boukamp, B. A.; van den Berg, A.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 1998, 120, 4652. (43) Beulen, M. J.; Kastenberg, M. I.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Langmuir 1998, 14, 7463. (44) Gorman, C. B.; Miller, R. L.; Chen, K.-Y.; Bishop, A. R.; Haasch, R. T.; Nuzzo, R. G. Langmuir 1998, 14, 3312. (45) Maeda, Y.; Yamamoto, H.; Kitano, H. J. Phys. Chem. 1995, 99, 4837. (46) Yoshizumi, A.; Kanayama, N.; Maehara, Y.; Ide, M.; Kitano, H. Langmuir 1999, 15, 482. (47) Maeda, Y.; Kitano, H. J. Phys. Chem. 1995, 99, 487. (48) Yamamoto, H.; Maeda, Y.; Kitano, H. J. Phys. Chem. B 1997, 101, 6855. (49) Maeda, Y.; Fukuda, T.; Yamamoto, H.; Kitano, H. Langmuir 1997, 13, 4187. (50) Fukuda, T.; Maeda, Y.; Kitano, H. Langmuir 1999, 15, 1887. (51) Kitano, H.; Taira, Y.; Yamamoto, H. Anal. Chem. 2000, 72, 2976. (52) Kitano, H.; Taira, Y. Langmuir 2002, 18, 5835.

Materials. The iniferter (Cys-BDC, Scheme 1) was prepared as described earlier.21 2-Methacryloyloxyethyl D-glucopyranoside (MEGlc, a mixture of R and β anomers (2.3:1)) was kindly donated by Nippon Fine Chemicals, Osaka, Japan.55-57 Methacrylic acid (MA) from Wako Pure Chemicals, Osaka, Japan, and N,Ndimethylacrylamide (DMAA) from Kohjin, Tokyo, Japan, were distilled in vacuo. 3-Aminopropyltriethoxysilane was purchased from ShinEtsu Chemicals, Tokyo, Japan. Other reagents were also obtained from commercial sources. A micro cover glass was purchased from Matsunami Glass, Osaka, Japan. All aqueous solutions and sensor chips were prepared with ultrapure water (18 MΩ cm-1, Millipore System). Preparation of SAM-Forming Polymer (Scheme 1). CysBDC (8.5 mg), MEGlc (50% (w/w) aqueous solution, 1.5 mL), and N,N,N′,N′-tetraethylthiuram disulfide (TD, capping reagent, 7.5 mg) were dissolved in anhydrous tetrahydrofuran (THF) and, after passing N2 gas for several minutes, photoirradiated in a quartz cell with a high-pressure mercury lamp (UI-501C, 250 W, Ushio, Tokyo, Japan) from a distance of 25 cm (3.0 × 1014 quanta s-1) at 25 °C for 30 min. The PMEGlc-carrying polymer was purified by ultrafiltration (membrane, Amicon YM1; exclusion limit, 103) and finally lyophilized in the dark (PMEGlc, Polymer1, 0.42 g; Table 1). At the purification of Polymer-3, a fraction between 103 and 104 Da was collected using YM1 and YM10 (exclusion limit, 104) membranes. Polymers composed of different monomer residues were prepared using the same procedure (Table 1). The 1H NMR measurements definitely showed the presence of aromatic protons, which are solely ascribable to the iniferter moiety, in all the polymers prepared by the Cys-BDC iniferter. The molecular weights of the polymers were determined by gel-permeation chromatography (GPC) using pullulan as standards (column, Wako Gel G-50, Wako Pure Chemicals; mobile (53) Kitano, H.; Saito, T.; Kanayama, N. J. Colloid Interface Sci. 2002, 250, 134. (54) Kitano, H.; Ohhori, K. Langmuir 2001, 17, 1878. (55) Kitano, H.; Ohno, K. Langmuir 1994, 10, 4131. (56) Kobayashi, K.; Kakishita, N.; Okada, M.; Akaike, T.; Kitazawa, S. Makromol. Chem., Rapid Commun. 1993, 14, 55. (57) Kitano, H.; Maehara, Y.; Matano, M.; Sugimura, M.; Shigemori, K. Langmuir 1997, 13, 5041.

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Table 1. Characteristics of Various Prepared Polymers polymer Polymer-1 Polymer-2 Polymer-3 PDMAA PMA

Cys-BDC (mg) 8.5 15.0 21.8 37.2 17.4

solvent (mL)

monomer (mL)

2.5 2.5 2.5 2.0 3.2

1.50b 0.70b 0.50b 0.52 0.84

irradiation time (min) 30 150 180 240 300

yield (g)

Mw (DP)a

Mw/Mn

0.42 0.17 0.11 0.07 0.29

5.0 × 1.7 × 104 (57) 7.6 × 103 (24) 2.2 × 103 (19) 1.4 × 103 (14)

1.32 2.58 1.90 1.43 1.09

104 (171)

a Molecular weight and degree of polymerization of each polymer chain. The total M of the prepared polymer was twice this value. b 50% w (w/w) aqueous solution.

Chart 1. Probable Structure of the Prepared Biofunctionalized Sensor Chip

phase, 0.1 M NaBr) and matrix-assisted laser desorptionionization time-of-flight mass spectrometry (in the case of poly(methacrylic acid) (PMA)) (MALDI-TOF, Voyager RP, PerSeptive Biosystems). The Mn values for these polymers estimated by 1H NMR were roughly in agreement with those determined by GPC. Preparation and Characterization of Gold Colloids. All glassware used for preparation of the colloids was thoroughly washed with aqua regia (3:1 HCl-HNO3) and rinsed extensively with water. Gold colloids were prepared by the reduction of HAuCl4 with sodium citrate in water at 100 °C as previously reported.58 The average hydrodynamic diameter of the gold colloids was estimated to be 40 nm by the dynamic light scattering technique (DLS-7000, Otsuka Electronics, Hirakata, Osaka, Japan; light source, He-Ne laser at 632.8 nm). Fixation of the Colloidal Gold Monolayer on the Glass Substrate. A cover glass (thickness, 160 µm) cut into 9.5 × 24 mm pieces was used for the fabrication of the colloidal Au monolayer. The glass substrates were cleaned by sonication for 2 h in water containing a 3% detergent (Contaminon N, Wako Pure Chemicals) and washed thoroughly with water. The substrates were further cleaned in concentrated HNO3 for 4 h, washed thoroughly with water, and, after cleaning using sonication for 2 min, dried in a drying oven at 70 °C overnight. The substrates were immersed in a 10% (v/v) 3-aminopropyltriethoxysilane aqueous solution for 20 min, rinsed with water and sonicated for 20 s, and finally dried at 70 °C for 3 h. The amineterminated glass coverslips were subsequently immersed into a colloidal gold solution overnight to form a self-assembled monolayer of Au colloids. Functionalization of the Colloidal-Gold-Fixed Glass Substrate (Chart 1). The colloidal-Au-fixed glass substrates were modified with various disulfide-carrying polymers prepared by immersion in a 0.5 mg/mL polymer solution for 4 h and subsequent extensive rinsing with water. The SAM-carrying glass substrates were stored in water until the absorption measurements. Absorption Measurements. A UV-visible-near-infrared spectrophotometer (Lambda 19 UV/VIS/NIR spectrometer, Perkin-Elmer) was used to measure the absorbance of the immobilized Au colloids on a glass substrate placed in a quartz cell (light path length, 10 mm). Spectra of the device were obtained in transmission mode over a range of 350-850 nm at 25 °C (58) Kanayama, N.; Kitano, H. Langmuir 2000, 16, 577.

(controlled by a Peltier device). In the experiments using concanavalin A (Con A), a HEPES (N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid, 5 mM, pH 8.0, [MnCl2] ) [CaCl2] ) 0.1 mM) buffer was used, while in the experiments using bovine serum albumin (BSA) and egg white lysozyme, a phosphate buffer (10 mM, pH 7.0) was used. Atomic Force Microscopy (AFM) Images. The immobilized Au colloids on the glass substrate were imaged by an atomic force microscope (Multimode Nanoscope, Seiko Instruments, Tokyo, Japan) in tapping mode in air using a standard Si2N4 cantilever.

Results and Discussion Construction of the Polymer SAM on the Gold Colloid. Previously, we first deposited a Cys-BDC SAM on the two-dimensional Au surface and tried to UV irradiate to the iniferter SAM in the presence of MA.54 However, the formation of PMA on the Au surface following photoirradiation could not be confirmed by the increase in potential difference (∆Ep, difference between peaks of oxidation and reduction) of the cyclic voltammograms of K3(CN)63-/4-. Furthermore, the ∆Ep value of the electrode photoirradiated with MA (64 mV) was smaller than that of the starting Cys-BDC SAM (81 mV) and close to that of the bare electrode (59 mV). These results showed that the iniferter SAM was mostly decomposed or forced to migrate from the gold surface during UV irradiation. Therefore, we changed the strategy to modification of the Au surface with the polymer-carrying disulfide, which had previously been prepared in the solution phase. In membrane systems (liposome, lipid monolayer, etc.), it has very often been reported that lectin does not bind to an incorporated sugar-carrying amphiphile with a short spacer group between the sugar residue and a hydrophobic anchor.55,59,60 This is due to a large steric hindrance preventing the huge lectin from approaching the small sugar residue on the membrane surface. Therefore, PMEGlc which has a long polymer chain with many pendent sugar groups was adopted as a “fly” to fish for a lectin in this study. The present strategy for the construction of the sensor chip based on the method mentioned above can be summarized as follows: At first, a pristine glass surface was functionalized by the formation of a 3-aminopropyltriethoxysilane SAM. Next, the amine-terminated glass surface was immersed in a dispersion of colloidal gold, which resulted in the formation of a monolayer of gold colloids on the glass surface. Finally, the colloidal-goldfixed glass substrate was immersed in a solution of a previously prepared functional polymer possessing a disulfide group. The self-assembled gold colloids on the glass and Polymer-1-modified sensor chip were characterized spectrophotometrically (Figure 1). According to the previous study,17 the aggregation of colloidal gold would result in the coupling of plasmons of individual particles and would be reflected in the UV-visible spectrum as a significant (59) Kawaguchi, T.; Tagawa, K.; Senda, F.; Matsunaga, T.; Kitano, H. J. Colloid Interface Sci. 1999, 210, 290. (60) Kitano, H.; Ishino, Y.; Yabe, K. Langmuir 2001, 17, 2312.

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Figure 1. Absorption spectrum of a colloidal Au dispersion and that of PMEGlc (Polymer-1)-modified Au colloids on a glass surface. Spectra were normalized to their absorption maxima. (a) Colloidal Au dispersion; (b) colloidal-Au-fixed glass substrate; (c) Polymer-1-immobilized Au colloid on a glass surface.

increase in the peak absorbance. The absence of such a feature in the UV-visible spectra of the glass covered with colloidal gold and the subsequently prepared Polymer1-modified sensor chip exhibited that the gold colloids were immobilized as a monolayer on the glass surface and were isolated from each other. Furthermore, to make sure the polymer synthesized by the Cys-BDC iniferter carries a disulfide bond (sulfur atom) and binds to gold via an Au-S bond, the Au-fixed glass substrate was immersed in a solution of the polymers without sulfur atoms (PMA and PMEGlc prepared only with a radical initiator, 2,2′-azobisisobutyronitrile (AIBN)), too. In this case, an increase of absorbance was not observed at all. These results imply that the polymer synthesized by the Cys-BDC iniferter was accumulated on the gold surface with the Au-S bond. The diameter of the Au colloids determined by dynamic light scattering (DLS) and AFM was ca. 40 nm, and the surface coverage of colloidal gold was estimated to be 5%. The gold colloids were isolated from each other in the AFM image, which is in agreement with the results in Figure 1. On the amine-terminated glass surface, arrangement of the colloidal gold can be controlled utilizing various interactions such as colloid-colloid interaction and colloid-amine interaction, effectively.17 In other words, by adjusting the size of the gold colloids before the contact with the amine-terminated glass substrate, the density of colloidal gold on the glass surface can be controlled. Furthermore, Kooij et al. reported the ionic strength dependence of gold nanoparticle assemblies analyzed in terms of radial distribution functions.61 They showed that the minimum interparticle separation for these particles with diameters in the low-nanometer range was tunable by varying the ionic strength of the solution from which the particles are deposited. However, the way to control surface density of the colloidal gold by changing ionic strength has a probability of flocculation in solution, and it should be executed very carefully. The optimization of the surface density of the colloidal gold as a sensing element is now examined from various viewpoints. Absorbance Changes with Refractive Index of the Surrounding Medium. Next, we examined the ability of the polymer-SAM-modified gold colloids on the glass substrate to induce changes in the refractive index of the surrounding medium appearing in the absorbance spectrum. Previously, Nath and Chilkoti13 reported that the absorbance spectrum of a colloidal-gold-covered glass substrate exhibited a red shift in the peak wavelength as a function of the refractive index of the solvent in the (61) Kooij, E. S.; Brouwer, E. A. M.; Wormeester, H.; Poelsema, B. Langmuir 2002, 18, 7677.

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range of 1.33-1.49. Our result also implied the linearity between the refractive index and the absorbance peak (λmax) or the absorbance at 550 nm. Templeton et al. showed that Mie theory predicts a red shift in the position of λmax and an increase in the absorbance maximum with an increase in refractive index of the medium,62 which is consistent with the present experimental observations. Stability of Sensor Chips. To examine the stability of the gold colloids, the salt-induced coagulation of the colloids was examined on a glass surface. By the addition of electrolyte, the stability of gold colloids was largely reduced because of the shielding of electrostatic repulsion between the colloid particles.63 The absence of effective electrostatic repulsion between the colloidal particles results in “rapid coagulation”, and the lowest electrolyte concentration to induce the rapid coagulation is known as the “critical flocculation concentration” (CFC), where the process is completely diffusion-controlled. The value of the CFC (electrolyte, sodium chloride) for the gold colloids examined in this work was 5.0 × 10-2 M. In contrast, for the gold-colloid-fixed glass surface, there was no significant change in the absorption spectrum after immersion in aqueous electrolyte solutions (even in 3 M sodium chloride), showing the enormous stability of the gold colloids. The high density of amino groups on the glass surface might lead to strong binding between the gold colloids and glass surface, because many amino groups could bind to each gold colloid particle. The sensor chip modified with the sugar-carrying polymer SAM gave the same results. The SAM-modified sensor chip may be more stable at high electrolyte concentration in comparison with a SAM-free gold-colloid-fixed glass substrate, due to the enormous steric stabilization effect of the PMEGlc layer stretching outward from the surface of the colloids. Colloids covered with polymer chains very often show no flocculation even at very high salt concentration.46 Recognition of Sugar Residues on Colloids by Lectin. Since the PMEGlc-modified colloidal Au on the glass surface can be used in a buffer solution, we examined the recognition of sugar residues in the PMEGlc SAM by Con A using the UV-visible spectrophotometer. After the Polymer-1-modified sensor chip was immersed in a Con A solution, an increase appeared in the absorption at 550 nm with a concomitant red shift in the peak absorbance. Figure 2 shows the absorbance spectra of the Polymer1-modified sensor chip before and after immersion in the Con A solution. Since glucose residue is well-known to be bound by Con A,64 the increase in the absorbance change can be attributed to the binding of sugar residues in the polymer by a tetrameric protein, Con A. To confirm this, the effect of R-methyl D-mannopyranoside (R-MeMan), which has a stronger affinity than glucose for binding to Con A, was examined. The absorption spectrum changed to the same spectrum as that of the sample before the immersion in the Con A solution. The strong inhibitory effect of R-MeMan showed that the absorbance change is due to the specific recognition of glucose residues in the polymer by Con A. Furthermore, the inset shows repeated usability of the Polymer-1modified sensor chip for the binding of Con A after immersion in a freshly prepared R-MeMan solution. Kinetics of Adsorption and Desorption of Con A from the SAM-Modified Sensor Chip. We further (62) Templeton, A. C.; Pietron, J. J.; Murray, R. W.; Mulvaney, P. J. Phys. Chem. B 2000, 104, 564. (63) Everett, D. H. In Basic Principles of Colloid Science; Royal Society of Chemistry: London, 1988. (64) Farina, R. D.; Wilkins, R. G. Biochim. Biophys. Acta 1980, 631, 428.

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relaxation process using eqs 1 and 2.57,65

∆Absassoc ) Abst - Abs0 ) [Con A]kassoc∆Absmax{1 - exp[-([Con A]kassoc + kdiss)t]} [Con A]kassoc + kdiss

(1) ∆Absdiss ) Abst - Abstf∞ ) (Abs0 - Abstf∞) exp(-kdisst) + Abstf∞ (2)

Figure 2. Upper panel: absorbance of the glass chip before and after immersion in a Con A solution. Dashed line, before immersion in Con A solution. Solid line, after immersion in Con A solution. Dotted line, after further immersion in R-MeMan solution. [Con A] ) 0.2 mg/mL in buffer solution (pH 8.0, 5 mM HEPES, 0.1 mM CaCl2, and MnCl2). [R-MeMan] ) 10 mM in HEPES buffer solution. Lower panel: (A) before immersion in Con A solution, (B) after immersion in Con A solution, (C) after further immersion in R-MeMan solution.

Figure 3. The time evolutions of absorbance for the Polymer1-immobilized sensor chip after immersion in various solutions: (a) Con A; (b-d) Con A and R-MeMan, lysozyme, and BSA. Lines b and c mostly overlapped. The Con A solution was changed to freshly prepared R-MeMan solution at the dashed arrow. [Con A] ) [BSA] ) [lysozyme] ) 0.2 mg/mL, [R-MeMan] ) 10 mM.

investigated the kinetics of Con A adsorption by monitoring the absorbance change at 550 nm in real time. As shown in Figure 3, the time dependence of the specific binding of Con A could be followed using this sensor chip. Upon immersion in Con A solution, the absorbance at 550 nm for the Polymer-1-modified colloidal gold on the glass chip gradually increased and leveled off after ca. 30 min. Furthermore, when the sensor chip was incubated in the Con A solution beforehand and then immersed in R-MeMan, the absorbance at 550 nm slowly decreased. In contrast with Con A, BSA and egg white lysozyme showed only a very slight absorption change (Figure 3). The effect of specific binding of Con A on the absorption change is much greater than that of nonspecific adsorption of proteins, indicating that the nonspecific adsorption of the protein had little or no effect on the sensor chips. Therefore, it can be disregarded. Next, we determined the association and dissociation rate constants (kassoc and kdiss, respectively) from each

where ∆Absassoc and ∆Absdiss are the absorption changes in the association and dissociation processes at time t. Abs0, Abst, Abstf∞ are the absorbances at time ) 0, t, and infinity, respectively. ∆Absmax is the capacity of the adsorbed PMEGlc to bind Con A expressed in response units. These equations are applicable under the assumption that each sugar residue on the polymer SAM surface interacts with a lectin independently. Therefore, the amount of polymer attached and the amount of sugar residue on the surface have nothing to do with the association or dissociation rate constant (these equations do not have a term for the amount of polymer modified or the amount of sugar residue on the surface). The kassoc and kdiss values obtained from Figure 3 were 2.0 × 103 M-1 s-1 and 4.1 × 10-3 s-1, respectively. The apparent association constant (kassoc/kdiss) was 5.0 ( 0.2 × 105 M-1, which is substantially larger than that for the complexation of Con A with small sugar derivatives (RMeMan, (7.6 ( 0.2) × 103 M-1; methyl R-D-glucopyranoside, (2.4 ( 0.1) × 103 M-1),66 probably because of multipoint (cooperative) fixation of Con A by the glucose residues in Polymer-1 (the so-called “cluster effect”),67 which might significantly decelerate the dissociation. As mentioned above, eqs 1 and 2 assumed a 1:1 association of Con A with a small molecular weight sugar (glucose or mannose). The association constant (kassoc/kdiss) estimated using these equations was much larger than that for small sugars determined by microcalorimetry (four small sugars independently bind to a Con A molecule). This difference can be ascribed to a collaboration of neighboring sugar residues in the polymer SAM at the binding to Con A, which implies that there is a cluster effect without a doubt. Previously, we examined the association and dissociation of Con A with a polymer brush composed of A-B-A block polymer (PMEGlc-b-PSt-b-PMEGlc, PSt is polystyrene) on a poly(methyl methacrylate) waveguide using a multiple internal reflection fluorescence method.57 The association constant obtained (2.0 × 106 M-1) was larger than that obtained in this work, probably due to the presence of the N,N-diethyldithiocarbamoyl group which made the end of the polymer brush more hydrophobic, resulting in a partly shrunken conformation. Adsorption of three kinds of proteins (Con A, BSA, and lysozyme) to various SAMs (PMA and PDMAA) was also examined. Since the absorbances (at 550 nm) of each SAMmodified sensor chip differed from each other, the differences in absorbance before and after the contact of proteins with the SAMs of PMA and PDMAA were compared. In the case of the PDMAA SAM, the increase in absorbance for the adsorption of Con A, BSA, and lysozyme to the SAM was negligible, showing a very low (65) Lookene, A.; Chevreuil, O.; Østergaard, P.; Olivecrona, G. Biochemistry 1996, 35, 12155. (66) Weatherman, R. V.; Mortell, K. H.; Chervenak, M.; Kiessling, L. L.; Toone, E. J. Biochemistry 1996, 35, 3619. (67) Lee, R. T.; Lee, Y. C. Carbohydr. Res. 1974, 37, 193.

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Morokoshi et al.

In Figure 4, absorption changes for two kinds of polymermodified sensor chips are shown (Polymer-1, DP ) 171; Polymer-2, DP ) 57). There seemed to be no significant difference between the data, suggesting that the polymers accumulated on the Au colloid are long enough to realize a cluster effect, which resulted in similar very high sensitivity. There is a possibility that since the disulfide group is located in the center of the polymers, the portion with a smaller molecular weight for each polymer can be relatively more easily chemisorbed to the Au colloid, which apparently diminishes the effect of molecular weight. Therefore, absorption changes of the Polymer-3 (DP ) 24)-carrying sensor chip were followed to clarify the effect of the polymer length on the detection limit of Con A. The detection limit increased to 19 nM (data not shown), due to the increase in steric hindrance for Con A to approach sugar residues in the polymer SAM. The difference in detection limits for the sensor chips carrying Polymer-2 and Polymer-3 suggests the presence of a critical value in chain length for the effective detection of a target molecule by the polymer-modified sensor chips examined here. Figure 4. Time dependence of surface plasmon absorbance at 550 nm after immersion of the glass plate modified with the PMEGlc-carrying colloidal gold in Con A solution and absorbance change as a function of Con A concentration. Upper panel (Polymer-1 SAM): (a-g) [Con A] ) 9.6 × 10-6, 1.9 × 10-6, 1.9 × 10-7, 1.9 × 10-8, 1.9 × 10-9, 1.9 × 10-10, and 1.9 × 10-11 M. Lower panel (Polymer-2 SAM): (a-f) [Con A] ) 9.6 × 10-6, 1.9 × 10-6, 1.9 × 10-7, 1.9 × 10-8, 1.9 × 10-9, and 1.9 × 10-10 M.

nonspecific adsorption of the proteins. On the other hand, in the case of the PMA SAM, the absorbance after contact with lysozyme increased significantly by comparison with that for BSA and Con A. Electrostatic interactions (attraction or repulsion) between the protein (lysozyme pI ) 10.5-11.0, BSA pI ) 4.7-4.9, and Con A pI ) 4.55.5)68 and the negatively charged PMA surface at pH 7 played an important role in this case. Finally, the detectability of the Con A sensor was estimated using various concentrations of Con A. Figure 4 shows that the maximum amount of Con A bound to the surface in the steady state is directly related to the solution concentration, and an absorbance change at 550 nm could only be observed when the solution concentration was above 1.9 nM. The very small detection limit of Con A using the Polymer-1-carrying sensor chip (1.9 nM) indicates that this sensor chip has a very high relative sensitivity. (68) In Biochemistry Handbook; Yamakawa, T., et al., Eds.; Tokyo Kagaku Dojin: Tokyo, 1979.

Conclusion The disulfide-carrying PMEGlc prepared by the iniferter technique could easily be accumulated on colloidal Au attached to a glass substrate. The changes in the absorbance spectrum associated with the specific binding of Con A to the PMEGlc SAM on the Au colloids could easily be measured using a UV-visible spectrometer, which is a quite efficient way to quantify the interactions. The polymer-coated sensor chip had very high stability and detectability ([Con A] ) 1.9 nM), while it could be repeatedly used to detect lectin. Acknowledgment. We are thankful to Dr. Paul B. Davies, Department of Chemistry, University of Cambridge, for his invaluable suggestions in the preparation of the paper. We are indebted to Professor H. Shinohara of this department for the AFM measurements. We are grateful to Mr. Takayuki Mori of this laboratory for the GPC measurements. We wish to thank Nippon Fine Chemicals for the gift of MEGlc. Supporting Information Available: (1) DLS data of the colloidal Au and photos of the colloidal-Au-fixed glass substrate taken by AFM. (2) Absorbance spectra of Polymer-1modified Au colloids on glass immersed in various solvents. (3) Absorbance change of the sensor chip after contact of proteins (BSA, Con A, and lysozyme) with the PMA SAM. This material is available free of charge via the Internet at http://pubs.acs.org. LA049201X