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Ligand Density Effect on Biorecognition by PEGylated Gold Nanoparticles: Regulated Interaction of RCA120 Lectin with Lactose Installed to the Distal End of Tethered PEG Strands on Gold Surface Seiji Takae,† Yoshitsugu Akiyama,† Hidenori Otsuka,‡ Teisaku Nakamura,† Yukio Nagasaki,§ and Kazunori Kataoka*,†,| Department of Materials Science and Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, Biomaterials Center, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan, Tsukuba Research Center for Interdisciplinary Materials Science, Tsukuba University, Tennoudai 1-1-1, Tsukuba 305-8573, Japan, and Center for Disease Biology and Integrative Medicine, School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received September 14, 2004; Revised Manuscript Received November 17, 2004
PEGylated gold nanoparticles (diameter: 20 nm) possessing various functionalities of lactose ligand on the distal end of tethered PEG ranging from 0 to 65% were prepared to explore the effect of ligand density of the nanoparticles on their lectin binding property. UV-visible spectra of the aqueous solution of the nanoparticles revealed that the strong steric stabilization property of the PEG layer lends the nanoparticles high dispersion stability even under the physiological salt concentration (ionic strength, I ) 0.15 M). The number of PEG strands on a single particle was determined to be 520 from thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) observation under controlled acceleration voltage revealed the thickness of the PEG layer on the nanoparticle to be ∼7 nm. The area occupied by a single lactose molecule on the surface of PEGylated gold nanoparticles was then calculated based on TGA and SEM results and was varied in the range of 10-34 nm2 depending on the lactose functionality (65∼20%). PEGylated gold nanoparticles with 40% and 65% lactose functionality showed a selective and time-dependent aggregation in phosphate buffer with the addition of Ricinus communis agglutinin (RCA120) lectin, a bivalent galactosespecific protein. The aggregates can be completely redispersed by adding an excess amount of galactose. Time-lapse monitoring of UV-visible spectra at 600-750 nm revealed that the aggregation of PEGylated gold nanoparticles was accelerated with an increase in both RCA120 concentration in the solution and the lactose density of the nanoparticles. Furthermore, the sensitivity of lectin detection could be controlled by the regulation of lactose density on the particle surface. Interestingly, there was a critical lactose density (>20%) observed to induce detectable particle aggregation, indicating that the interaction between the particles is triggered by the multimolecular bridging via lectin molecules. Introduction Nanometer-sized metal and semiconductor particles exhibit quantum size effects and have recently attracted growing attention as a simple and sensitive assay method of biologically relevant molecules through the conjugation of ligands on their surface.1 Particularly, ligand-conjugated gold nanoparticles, an aqueous solution of which is red in color due to the surface plasmon absorption around 520 nm, are useful for the simple and sensitive bioassays2 because their biospecific aggregation in aqueous medium leads to the formation of a new absorption band at longer wavelength due to the electric dipole-dipole interaction and the coupling * To whom correspondence should be addressed. Telephone: +81-35841-7138. Fax: +81-3-5841-7139. E-mail:
[email protected]. † School of Engineering, The University of Tokyo. ‡ National Institute for Materials Science (NIMS). § Tsukuba University. | School of Medicine, The University of Tokyo.
between the plasmons of neighboring particles.3 Nevertheless, the sensitivity of gold nanoparticles as a colloidal sensor system in biological fluids is impaired due to the nonspecific agglomerization particularly at the physiological condition with 0.15 M NaCl as well as to the nonnegligible adsorption of biological components such as proteins and DNA.4 Several methods have been applied so far to improve the dispersivity of gold nanoparticles in aqueous medium utilizing surfactants and polymer additives.5,6 Nevertheless, the long-term stability of the nanoparticles under physiological conditions (I ) 0.15 M) to avoid nonspecific aggregation has not been well characterized yet. An alternative approach to acquire sterically stabilized gold nanoparticles is, as we reported previously, to modify their surface with endfunctionalized and hydrophilic polymer strands such as heterobifunctional poly(ethylene glycol) (PEG) containing both mercapto and acetal terminal groups (acetal-PEG-SH).7 These R-acetal-PEGylated gold nanoparticles indeed ac-
10.1021/bm049427e CCC: $30.25 © 2005 American Chemical Society Published on Web 01/21/2005
Ligand Density Effect on Biorecognition
complished a high dispersion stability in physiological conditions. Note that an acetal moiety on the periphery of the PEG brushed-layer can easily be converted under gentle acidic condition into a reactive aldehyde group to conjugate various ligands. As a model ligand, lactose was successfully introduced in the distal end of the PEG chains, inducing selective and reversible aggregation of lactose-functionalized PEGylated gold nanoparticles with a concomitant color change (red-purple-red) by the addition of RCA120 lectin.8 The molecular recognition process responsible for the aggregation of ligand-installed PEGylated gold nanoparticles is induced through multivalent interaction bridged by the analyte molecules, e.g. lectin. As ligand density is an important factor in this process,6,8 it is essential to reveal the relationship between the sensitivity of the PEGylated gold nanoparticles and their ligand density for applying them in highly sensitive bioassay. Nevertheless, as far as we know, there is no systematic study on the stringent control of ligand density on gold nanoparticles surfaces to assess the multivalent interaction, which definitely contributes to an increased sensitivity and selectivity because the binding constant based on the multivalent interaction is several orders of magnitude higher than that based on only the monovalent interaction.9 In this regard, a study reported by Fantuzzi et al. is interesting, revealing that N-methylimidazole-fuctionalized gold nanoparticles behave as multivalent ligands for porphyrin arrays with an increase in binding constant of up to 3 orders of magnitude with respect to a monovalent system.10 The purpose of the study reported here is to reveal the ligand density effects on the aggregation behavior of ligandinstalled PEGylated gold nanoparticles through the evaluation of the interaction between lectin and lactosyl-PEGylated gold nanoparticles with regulated density of lactose. Here, a novel approach is introduced to regulate lactose density on PEGylated gold nanoparticles: Immobilization of the mixtures of oxidized dimers of acetal-PEG-SH and lactosyl-PEG-SH [(acetal-PEG-S-)2 and (lactose-PEG-S-)2, respectively] in various molar ratios. In this way, PEGylated gold nanoparticles with a systematically varying lactose density were prepared to explore the effects of ligand density on the aggregation behavior of gold nanoparticles induced by RCA120 lectin. Experimental Section Materials. p-Aminophenyl β-D-lactopyranoside (Toronto Research Chemicals, Toronto, Canada), sodium cyanoborohydride (Sigma-Aldrich Co., St. Louis, MO), gold colloid (diameter; 20 nm, British Biocell International, Cardiff, U.K.), bovine serum albumin (BSA, Sigma-Aldrich Co., St. Louis, MO), and Ricinus communis agglutinin (RCA120, Honen Co., Tokyo, Japan) and the other chemicals were used as received. Water was purified with a Milli-Q instrument (Millipore, Bedford, MA). Synthesis of (Acetal-PEG-S-)2 and (Lactose-PEG-S-)2. Synthesis of poly(ethylene glycol) derivatives containing both acetal and mercapto terminals (acetal-PEG-SH) was described elsewhere.7 To obtain the heterobifunctional PEG dimer (acetal-PEG-S-)2, 460 mg of acetal-PEG-SH was
Biomacromolecules, Vol. 6, No. 2, 2005 819 Scheme 1. Synthetic Route of (Lactose-PEG-S-)2
oxidized in 20 mL of DMSO/MeOH (1:1) mixture by stirring 24 h at room temperature in open air condition as shown in Scheme 1. Purification was carried out by dialysis against distilled water and then freeze-dried [yield: 74% (340 mg)]. The successive conversion of acetal groups at the end of (acetal-PEG-S-)2 (50 mg) to the aldehyde group [(CHOPEG-S-)2] was carried out by the addition of an acetic acid/ distilled water (10:1) mixture (3.3 mL). After 5 h stirring at 40 °C, the reaction mixture was added to 30 mL of chloroform followed by washing with 20 mL of saturated NaCl aqueous solution several times. The organic phase was then separated and dried with MgSO4. After the evaporation of the solvent, 30 mL of benzene was added and freezedried to obtain the white powder. Then, a 5-fold molar amount of p-aminophenyl β-D-lactopyranoside (20 mg) to aldehyde residue in 15 mL of 50 mM phosphate buffered solution (pH ) 7.0) was added to the (CHO-PEG-S-)2 in the same flask. After stirring for 15 h at room temperature, 3.0 mg of NaBH3CN (5-fold molar amount of aldehyde residue in this system) was added to the mixture and stirred for an additional 2 days. Purification was carried out by dialysis against distilled water overnight and then freezedried (yield: 78% (39 mg)). From gel permeation chromatography (GPC), the numberaveraged molecular weight (Mn) and the molecular weight distribution (MWD) of (acetal-PEG-S-)2 were determined to be 12 000 and 1.08, respectively, and Mn and MWD of (lactose-PEG-S-)2 were determined to be 12 200 and 1.08, respectively. Lactose conversion ratio was then determined to be 65% from the 1H NMR spectrum (Supporting Information, Figure S1) based on the peak intensity ratio of the phenyl protons of the conjugated p-aminophenyl β-Dlactopyranoside (6.5∼6.9 ppm) moiety to the methylene protons of PEG chain (3.3∼3.8 ppm). Preparation of PEGylated Gold Nanoparticles Possessing Varying Lactose Functionalities. To obtain PEGylated gold nanoparticles with varying lactose functionalities of 0, 20, 40, and 65% (abbreviated as lac0, lac20, lac40, lac65, respectively), 10 mL of the mixed solution of (acetal-PEGS-)2 and (lactose-PEG-S-)2 in different molar ratios (total concentration 20 µg/mL) was added to 5 mL of an aqueous dispersion of commercial gold nanoparticles (diameter ) 20 nm, 7.0 × 1011 particles/mL) at room temperature. After stirring overnight, to remove excess polymer from the solution, two repeated centrifugation (42 000 × g) and
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redispersion steps were performed. Finally, the PEGylated nanoparticles were suspended in 10 mL of phosphate buffered solution (PBS) (pH ) 7.4, I ) 0.15 M) containing 0.05 wt % bovine serum albumin (BSA). The dispersion stability of the PEGylated gold nanoparticles and their lectin-induced aggregation were evaluated spectrophotometrically by UV-visible spectrophotometer (V-550 UV/VIS spectrophotometer, JASCO, Japan) at 25 °C. The normalized integrated absorbance (NIA)11 between 600 and 750 nm was used as an indicator of the aggregate formation. The NIA is defined as follows: NIA(t) ) (At - A)/A where A is the initial integrated absorbance between 600 and 750 nm, and At is the integrated absorbance between 600 and 750 nm t hours after the addition of RCA120. Scanning Electron Microscopic (SEM) Measurements. To estimate the thickness of the PEG layer on the surface of the PEGylated gold nanoparticles, SEM images were taken at different acceleration voltages (4.7 and 1.0 kV) with a LEO 1550 electron microscope (Carl Zeiss, Germany). SEM samples were prepared by mounting a drop of the solution on carbon-coated Cu grids and allowing them to dry in the air. Image analyses to obtain average particle size and the size distributions were performed on a Macintosh Power Mac G4 computer using the public domain NIH Image program.12 Results and Discussion Preparation of PEGylated Gold Nanoparticles with Varying Lactose Functionality. As shown in Scheme 1, the PEG dimer (acetal-PEG-S-)2 (Mn ) 12 000, MWD ) 1.08) was prepared by a dimerization of acetal-PEG-SH through disulfide bonding in DMSO/MeOH. Acetal groups at both ends of (acetal-PEG-S-)2 were then transformed into aldehyde groups by a gentle acid treatment, followed by the reaction with p-aminophenyl β-D-lactopyranoside in the presence of NaBH3CN to undergo a reductive amination. The lactose conversion ratio of the obtained polymer, (lactosePEG-S-)2 (Mn ) 12 200, MWD ) 1.08), was determined to be 65% based on the peak intensity ratio of the phenyl protons of the conjugated p-aminophenyl β-D-lactopyranoside (6.5∼6.9 ppm) moiety to the methylene protons of the PEG chain (3.3∼3.8 ppm) in the 1H NMR spectrum (Supporting Information, Figure S1). Then, the mixtures of PEG dimers, (acetal-PEG-S-)2 and (lactose-PEG-S-)2, in varying molar ratios were reacted with commercially available gold nanoparticles (diameter: 20 nm) at room temperature in distilled water overnight. To remove excess polymer in the solution, repeated centrifugation (42 000 × g) and redispersion were carried twice. Finally, the PEGylated nanoparticles were suspended in PBS (pH ) 7.4, I ) 0.15 M) containing 0.05 wt % bovine serum albumin (BSA). Dispersion Stability of PEGylated Gold Nanoparticles. Availability of the PEG dimer, (acetal- or lactose-PEG-S)2, as effective PEGylation reagents of gold nanoparticles was confirmed from the redispersibility of the nanoparticles after centrifugation as well as from a profile of UV-visible
Figure 1. Dispersion stability of PEGylated gold nanoparticles in PBS (pH ) 7.4, I ) 0.15 M) containing several concentration of bovine serum albumin (BSA), (b) 0 µg/mL; (2) 50 µg/mL; (9) 200 µg/mL; (() 500 µg/mL.
spectra. PEGylated gold nanoparticles spun down at 42 000 × g for 30 min at 20 °C can be readily resuspended in deionized water as well as in PBS (pH ) 7.4, I ) 0.15 M). On the other hand, most of the bare gold nanoparticles were unable to be resuspended after the same centrifugation process. Then, the colloidal stability of (acetal-PEG-S-)2coated gold nanoparticles (lac0) was evaluated under the physiological salt condition (pH ) 7.4, I ) 0.15 M) by monitoring the change in the metachronic absorbance in the UV-visible spectrum. An aqueous solution of dispersed gold nanoparticles is red due to the surface plasmon absorbance centered around 523 nm in the optical spectrum. On the other hand, particle aggregation leads to the formation of a new absorption band at longer wavelengths than 600 nm.3 Dispersion of lac0 in PBS (pH ) 7.4, I ) 0.15 M) indeed maintained an absorption maximum unchanged around 523 nm without any emergence of new absorption bands at larger wavelengths characteristic of aggregates. Nevertheless, there is a gradual decrease in the relative absorbance at 523 nm with time due to a decrease in the particle concentration resulting from the adsorption of the nanoparticles on the glass wall of the vessel. To prevent lac0 from adsorbing on the vessel wall, a small amount of bovine serum albumin (BSA) was added into the solution. As shown in Figure 1, the addition of 500 µg/mL (0.05 wt %) BSA completely inhibited this nonspecific adsorption of the nanoparticles to the vessel wall, allowing a highly stable dispersion of PEGylated gold nanoparticles in physiological salt condition to be obtained. Note that bare gold nanoparticles in PBS with 0.05 wt % BSA rapidly underwent aggregation to have an appreciable increase in the absorbance of around 650 nm (data not shown). Characterization of PEGylated Gold Nanoparticles Possessing Regulated Density of Lactose on the Distal End of PEG Strands. The number of PEG chains immobilized on a single gold nanoparticle was determined by thermogravimetric analysis (TGA). A clear decrease in the weight of lac0 was observed in the TGA curve in the range of 300∼400 °C due to the thermal decomposition of PEG moieties.13 Then, the number of PEG chains on each gold nanoparticle was calculated to be 520, using the concentration of gold nanoparticles (7.0 × 1011 particles/mL) provided by the manufacturer and the weight-loss due to PEG decom-
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Ligand Density Effect on Biorecognition Table 1. Number of PEG Chain and Ligand Density on Gold Nanoparticles Estimated from the Results of TGA and SEM lactose functionality (%)
20
40
65
number of lactose molecules on 1 particle area occupied by 1 lactose molecule (nm2)a average distance between lactose moieties (nm)b
104 34 5.8
208 17 4.1
338 10 3.2
a Surface area of PEGlyated gold nanoparticles (4π(33.3/2)2 nm2) was divided by number of lactose molecules on a single particle. b Square root of area occupied by 1 lactose molecule.
Figure 3. Change in the UV-vis spectra of lac65 with time after the addition of 200 µg/mL RCA120. Dotted line 0 h; thin solid line 1 h; dotted-dashed line 2 h; thick solid line 4 h; and dashed line the spectrum after the addition of excess D-galactose (10 mg/mL) to 4 h. The insert is the expanded spectra between 510 and 540 nm.
Figure 2. SEM images of PEGylated gold nanoparticles. The acceleration voltages are (a) 4.7 and (b) 1.0 kV, respectively.
position measured by TGA. Based on this calculation, the number of lactose molecules on a single gold nanoparticle was also determined as summarized in Table 1. Scanning electron microscopy (SEM) was used to estimate the thickness of the PEG layer of PEGylated gold nanoparticles. Figure 2, parts a and b, shows SEM images photographed with 4.7 and 1.0 kV acceleration voltages, respectively. Note that secondary electrons were detected strongly from gold atoms at a higher acceleration voltage (4.7 kV), whereas at a lower acceleration voltage (1.0 kV), secondary electrons from the PEG layer were also detected. The average diameter of PEGylated gold nanoparticles was then determined to be 33.3 ( 2.8 nm (mean ( SD, n ) 914) from the SEM images at 1.0 kV (Figure 2b) with NIH image software.12 On the other hand, the diameter determined from the image taken at 4.7 kV was consistent with that of bare
gold nanoparticles (20 nm) provided by the manufacturer. Consequently, the thickness of the PEG layer on the gold nanoparticle was estimated to be ∼7 nm. Note that the endto-end distances (R) of PEG [M ) 6000 g/mol, degree of polymerization (DP) ) 136] based on zigzag, meander,14 and random coil15 models were calculated to be 47.6, 24.527.2, and 5.9 nm, respectively. Thus, it is likely that PEG strands immobilized on gold nanoparticles may adopt a conformation of slightly stretched random coil. Consequently, lactose densities and the average distances between lactose moieties on lac20, lac40, and lac65 were calculated from SEM and TGA data and are summarized in Table 1. These density and distance values, calculated based on the SEM observation for dry samples, were assumed to be valid for the gold nanoparticles dispersed in the aqueous solution and were utilized to evaluate lectin-induced aggregation of the nanoparticles in the following section. Note that the dynamic light scattering (DLS) measurement of the aqueous dispersion of the PEGylated gold nanoparticles and the bare gold nanoparticles gave the hydrodynamic diameters of 40.9 and 25.1 nm, respectively. Then, the subtraction of the latter from the former yields the PEG thickness in the wet state as 7.9 nm, which is consistent with the value determined by SEM. This negligible discrepancy in the PEG thickness between dry and wet samples may presumably be due to the relatively high density of PEG brushes on the gold surface. Aggregation of PEGylated Gold Nanoparticles by RCA120 Lectin. Figure 3 shows a typical time-dependent change observed in UV-visible spectra of PEGylated gold nanoparticles possessing 65% lactose on the surface (lac65) after the addition of 200 µg/mL RCA120. The surface plasmon band underwent gradual red-shift from 523 nm as the growth of the aggregate occurred through the interaction of RCA120 with gold nanoparticles.3 After 4 h of incubation, macroscopic aggregates were eventually precipitated out from the solution. It is worth noticing that the spectrum underwent a
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Takae et al. Table 2. Assumed Number of Monovalently bound RCA120 Lectin Molecules Per Gold Nanoparticle at Equilibrium Condition (Nb)a lectin conc
10 µg/mL
25 µg/mL
50 µg/mL
100 µg/mL
lac20 lac40 lac65
0.46 0.92 1.5
1.1 2.3 3.7
2.3 4.5 7.3
4.4 8.8 14
a N is calculated from the equilibrium constant below, assuming the b monovalent lectin association with lactose on gold nanoparticles.
RCA120 + lactose h RCA120-lactose
Ka )
Figure 4. Time-dependent change in normalized integrated absorbance (NIA) of lactose-installed PEGylated gold nanoparticles after the addition of varying concentration of RCA120 (b, 10 µg/mL; 4, 25 µg/mL; 9, 50 µg/mL; (, 100 µg/mL; O, 200 µg/mL; 2, 400 µg/mL; 0, 800 µg/mL; ], 1000 µg/mL). (a) lac20; (b) lac40; and (c) lac65.
complete recovery to the initial profile after the addition of excess amount of galactose (10 mg/mL; Figure 3, dashed line), indicating a redispersion of these PEGylated gold nanoparticles due to the competitive binding of galactose to RCA120. This reversible aggregation-dispersion behavior was further ascertained by TEM (Supporting Information, Figure S2). Note that (acetal-PEG-S-)2-modified gold nanoparticles (lac0) exhibited no change in the spectrum even after the addition of RCA120 (Supporting Information, Figure S3), indicating that the lactose-installed PEGylated gold nanoparticles underwent lectin-specific aggregation with negligible nonspecific interaction. Since the lactose density on the surface is systematically changed for the samples (lac20, lac40, and lac65) depicted
[RCA120 - lactose] [RCA120][lactose]
) 2.67 × 104 M-1 (25 °C)
in Table 1, a relationship between the sensitivity of lectininduced aggregation and the lactose density was then evaluated in detail from the normalized integrated absorbance (NIA)11 between 600 and 750 nm defined in the Experimental Section. There was an obvious increase in NIA with time and lectin concentration dependent manner for lac40 and lac65, indicating the progressive aggregation of gold nanoparticles triggered by lectin (Figure 4, parts b and c). Although lac65 generally revealed higher response than lac40, both became insensitive in the concentration range of lectin lower than the critical value, 25 and 50 µg/mL for lac65 and lac40, respectively. This indicates the presence of critical lectin concentration as to the aggregation of gold nanoparticles. The average number of lectin molecules binding onto a single gold nanoparticle (Nb) at equilibrium condition was calculated, assuming the monovalent association, for varying lectin concentration from the association constant of RCA120 lectin and lactose (Ka ) 2.67 × 104 M-1 at 25 °C)16 and was summarized in Table 2. Note that the calculated number of bound lectin molecules in Table 2 is the assumed value based on the monovalent scheme, and thus, the actual number should take a substantially higher value than the calculated one in the region higher than the critical lectin concentration, above which the multivalent binding starts to occur, inducing the lectin-bridged aggregation of nanoparticles. Consequently, the values in Table 2 may only be actual ones in the region under the critical lectin concentration. What we would like to on focus here is that the calculated number of bound lectin molecules (Nb) on a single nanoparticle, based on the assumed monovalent interaction, takes a higher value than 3 in the range of lectin concentration inducing obvious aggregation of lac40 and lac65. The interaction mode may switch from monovalent to multivalent in this range, suggesting the formation of a stable interparticle bridge. Presumably, the binding of more than 3 lectin molecules per particle may be required for the formation of the network of gold nanoparticles large enough to form the macroscopic aggregates. Furthermore, NIA of lac65 and lac40 revealed a proportional increase to lectin concentration in the range of 50∼400 and 25∼200 µg/mL, respectively (Figure 5), indicating that simple quantification of lectin can be accomplished in the wide concentration range by the use of lactose-installed PEGylated gold nanoparticles with optimized lactose density. A drop of NIA at large RCA120 lectin concentration was due to the precipitation of aggregated gold nanoparticles and may not be caused by the saturation of the nanoparticle surface by singly occupied
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surface is more than 17 nm2.19 Presumably, lactose density on lac20 may not be high enough to form a multi-molecular bridging structure by RCA120. These results clearly indicate that the regulation of ligand density on the surface of gold nanoparticles is a crucial factor to control the sensitivity of gold colloidal sensors based on the biospecific aggregation. Conclusions
Figure 5. NIA obtained 2 h after the addition of RCA120 in varying concentration to the suspension of lactose-installed PEGylated gold nanoparticles (b, lac20; 9, lac40; 2, lac65). (a) logarithmic scale; (b) linear scale.
RCA120 molecules, considering the value of the association constant. Choice of smaller-sized particles with an appropriate ligand density may prevent the precipitation to permit a quantitative aggregation assay in a wider concentration range by the use of lac65 and lac40. On the other hand, lac20 did not undergo lectin-induced aggregation even in the range of lectin concentration (Figure 4a) enough to attain the Nb higher than 3 (>100 µg/mL). It is likely that there exists a critical lactose density on the gold nanoparticles to form lectininduced aggregates between 20% and 40% of lactose functionality. The area occupied by a single RCA120 lectin molecule estimated from its size17 is 24 () 6 × 4) nm2, whereas the area occupied by a single lactose molecule on the particle surface estimated from the number of lactose molecules and the surface area of nanoparticles are 34 and 17 nm2 for lac20 and lac40, respectively (Table 1). The binding constant of RCA120 with lactose is reported to be on the order of 104 M-1 (see Table 2) and is insufficiently low to form a stable network of lactose-installed gold nanoparticles bridged by a single lectin molecule with divalent functionality. It may be reasonable to assume that more than two lectin molecules may concomitantly participate in the stable interparticle bridge to enhance the association force through multivalent interaction. It is reported that lactose-installed polymeric micelles behave as multivalent ligands for lectins immobilized on a gold surface with an increase in the binding constant of up to 2 orders of magnitude with respect to a monovalent system.18 Furthermore, Woller and co-workers demonstrated the importance of multivalent interaction, showing that the recognition property of mannose/hydroxyl-functionalized dendrimers against Concanavalin A (Con A) lectin extremely decreased when the area per mannose molecule on the dendrimer
PEGylated gold nanoparticles possessing a regulated density of lactose (0∼65%) on the distal end of PEG were prepared by treating gold colloids with a mixed solution of (lactose-PEG-S-)2 and (acetal-PEG-S-)2 of varying compositions. The number of PEG strands on each particle and average diameter of PEGylated gold nanoparticles were estimated from the results of TGA and SEM measurements, respectively, allowing the lactose density on the surface of PEGylated gold nanoparticles with different lactose functionality to be determined. These PEGylated gold nanoparticles are highly stable under physiological condition, and eventually, those having 65% functionality of lactose (lac65) underwent sensitive RCA120 lectin-induced aggregation as well as quantitative re-dispersion with an addition of excess galactose. The shift in the plasmon absorption in UV-visible spectra due to the particle aggregation correlated with the lectin concentration, showing the feasibility of the system as a quantitative assay method. Furthermore, the concentration range of quantitative assay can be modulated by controlling the lactose density on the gold nanoparticles. A critical lactose density (>20%) to trigger lectin-induced aggregation was observed, suggesting that interaction between the nanoparticles may involve a multivalent recognition process. These findings indicate that the optimization of ligand density on the surface of gold nanoparticles as well as the prevention of nonspecific interactions through a stericrepulsive layer of PEG are key factors to construct highly sensitive colloidal biosensor based on the aggregation behavior of gold nanoparticles. Acknowledgment. The authors express their gratitude to Dr. Yuichi Yamasaki, University of Tokyo, for invaluable advice about image processing. This work was financially supported by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), by the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST) and by 21st Century COE Program “HumanFriendly Materials based on Chemistry” from MEXT. Supporting Information Available. 1H NMR spectrum of (lactose-PEG-S-)2, TEM images representing the reversible aggregation-dispersion behavior of PEGylated gold nanoparticles, and UV-visible spectra of lac0 after the addition of RCA120. These materials are available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. L. Anal. Chem. 2003, 75, 2377-2381. (b) Storhoff, J. J.; Marla, S. S.; Bao, P.; Hagenow, S.; Mehta, H.; Lucas, A.; Garimella, V.; Patno, T.; Buckingham, W.; Cork, W.; Mu¨ller, U. R. Biosens. Bioelectron.
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