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Notes Rapid, Quantitative Colorimetric Detection of a Lectin Using Mannose-Stabilized Gold Nanoparticles Duncan C. Hone, Alan H. Haines, and David A. Russell* School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom Received February 28, 2003
Molecular recognition events involving carbohydrates occur in a wide variety of important biological processes including the functioning of the immune system, interaction of viruses and bacteria,1 and tissue growth.2 Involved in these phenomena are cell-cell communication events, which are driven by highly specific carbohydratelectin interactions on opposing cell surfaces.3 The proteins involved in these molecular recognition processes are typically found in aggregated structures thereby presenting multiple binding or recognition sites for the carbohydrate ligand. Such aggregated structures enhance the low binding affinity of the monomeric carbohydrateprotein interaction, an observation referred to as the cluster glycoside effect.4 With consideration of the importance of such interactions, considerable research effort has focused on the development of new methodologies to study and quantify these molecular recognition events with particular attention on the formation of multivalent carbohydrate ligands. Previously, carbohydrate structures have been formulated: as self-assembled monolayers (SAMs) on 2D surfaces, both as single ligands5-7 and more recently within arrays;8-11 as dendrimers;12 within liposomes;13 and on the backbone of polymers.14 Recently, metal nanoparticles have been used to tether a variety of carbohydrate ligands.15-18 Such 3D multivalent ligands provide a globular structure on which clustering * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Karlsson, K. A. Curr. Opin. Struct. Biol. 1995, 5, 622-635. (2) Varki, A. Glycobiology 1993, 3, 97-130. (3) Sacchettini, J. C.; Baum, L. G.; Brewer, C. F. Biochemistry 2001, 40, 3009-3015. (4) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327. (5) Revell, D. J.; Knight, J. R.; Blyth, D. J.; Haines, A. H.; Russell, D. A. Langmuir 1998, 14, 4517-4524. (6) Kitov, P. I.; Railton, C.; Bundle, D. R. Carbohydr. Res. 1998, 307, 361-369. (7) Svedhem, S.; Ohberg, L.; Borrelli, S.; Valiokas, R.; Andersson, M.; Oscarson, S.; Svensson, S. C. T.; Liedberg, B.; Konradsson, P. Langmuir 2002, 18, 2848-2858. (8) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443-454. (9) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003, 19, 1522-1531. (10) Wang, D.; Liu, S.; Trummer, B. J.; Deng, C.; Wang, A. Nat. Biotechnol. 2002, 20, 275-281. (11) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2002, 41, 3583-3586. (12) Turnbull, W. B.; Kalovidouris, S. A.; Stoddart, J. F. Chem.s Eur. J. 2002, 8, 2988-3000. (13) Tagawa, K.; Sendai, N.; Ohno, K.; Kawaguchi, T.; Kitano, H. Bioconjugate Chem. 1999, 10, 354-360. (14) Choi, S.-K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 4103-4111. (15) Yoshizumi, A.; Kanayama, N.; Maehara, Y.; Ide, M.; Kitano, H. Langmuir 1999, 15, 482-488.
and orientation effects may be studied.16 In addition, the properties of the metal core can be applied to develop methods for the study of molecular recognition between carbohydrates and their respective binding proteins. For example, mannose-stabilized gold nanoparticles have been used to visualize FimH proteins on type 1 pili of Escherichia coli using electron microscopy.17 Other inherent properties which can be used are based on the optical characteristics of metal nanoparticles which are dominated by the coherent oscillations of the conduction band electrons induced by an interacting electromagnetic field.19 This absorption of light is known as surface plasmon absorption and is dependent on the dielectric properties of the metal, the size and shape of the particles, and the surrounding medium. Gold nanoparticles typically have a large surface plasmon absorption band centered at 520 nm, and thus aqueous solutions of gold nanoparticles appear red. Upon aggregation, the metal particles become closer in proximity and coupling interactions result in a shift in the surface plasmon absorption to lower energies. This concept has been utilized by Mirkin and co-workers to produce a colorimetric assay for the analysis of DNA.20,21 Subsequently this approach has been adopted for the detection of metal ions,22,23 the well-studied biotin-avidin interaction,24 and for antibody immunoassays.25 Additionally, Kataoka and co-workers have self-assembled poly(ethylene glycol)-derivatized carbohydrates for the colorimetric detection of Recinus communis agglutinin (RCA120).18 Here the further development of stabilized colloidal systems for the colorimetric detection of carbohydrate binding proteins is reported. A mannose derivative has been self-assembled onto preformed, citrate capped, watersoluble gold nanoparticles. Through the use of a short (C2) hydrocarbon tether between the gold surface and the mannose recognition center, a selective, quantitative, and, importantly, rapid colorimetric detection method has been developed for the carbohydrate binding protein concanavalin A. Experimental Section Reagents. All reagents were of analytical research grade and used without further purification. The lectins concanavalin A (Con A), isolated from jack bean (Canavalia ensiformis), and (16) de la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem., Int. Ed. 2001, 40, 2258-2261. (17) Lin, C.-C.; Yeh, Y.-C.; Yang, C.-Y.; Chen, C.-L.; Chen, G.-F.; Chen, C.-C.; Wu, Y.-C. J. Am. Chem. Soc. 2002, 124, 3508-3509. (18) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226-8230. (19) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: Berlin, 1995. (20) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (21) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (22) Lin, S.-Y.; Liu, S.-W.; Lin, C.-M.; Chen, C. Anal. Chem. 2002, 74, 330-335. (23) Obare, S. O.; Hollowell, R. E.; Murphy, C. J. Langmuir 2002, 18, 10407-10410. (24) Sastry, M.; Lala, N.; Patil, V.; Chavan, S. P.; Chittiboyina, A. G. Langmuir 1998, 14, 4138-4142. (25) Thanh, N. T. K.; Rosenzweig, Z. Anal. Chem. 2002, 74, 16241628.
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tetragonolobus purpureas, from asparagus pea, were purchased from the Sigma Chemical Co. Aqueous solutions were prepared using a 10 mM Tris stock at pH 7.6. Milli-Q water was used throughout for solution preparations. Synthesis of 2-Mercaptoethyl r-D-Mannopyranoside. The synthesis of 2-mercaptoethyl R-D-mannopyranoside was achieved as previously reported.5 Briefly, Fischer glycosidation of Dmannose with 2-bromoethanol, isolation of the resultant glycoside as its tetraacetate, and displacement of the bromide ion in this compound by treatment with potassium thioacetate gave the 2-acetylthioethyl mannoside tetraacetate, which on deesterification afforded the title glycoside. Synthesis of Modified Colloidal Gold Nanoparticles. Water-soluble gold nanoparticles were prepared according to the classical approach of Turkevich,26 using sodium citrate as both the reducing and capping agent. HAuCl4 (12.5 mg) was dissolved in 100 mL of water, and 50 mg of sodium citrate was dissolved in 50 mL of water; both solutions were then heated to 60 °C. Rapid addition of the sodium citrate solution to the gold solution was followed by increasing the temperature to 85 °C and continuous stirring for 2.5 h. A clear red solution was formed in which the particle concentration was approximately 3 nM. To 100 mL of the freshly prepared citrate-capped gold nanoparticle solution, 50 mg of the thiol-derivatized carbohydrate was added. Self-assembly was facilitated by leaving the solution for a period of 48 h. The mannose-stabilized nanoparticles were centrifuged (Beckman Avanti J-25 centrifuge) at 23 700g for 25 min. The modified particles were then resuspended in Tris buffer solution to give an approximate concentration of 10 nM. The centrifugation step was repeated three times to ensure complete removal of unbound mannose derivative. UV-Visible Absorption Measurements. A Hitachi U3000 UV-visible spectrophotometer was used to record both the absorption and kinetic spectra, at a temperature of 20 °C. Transmission Electron Microscopy (TEM) Measurements. Transmission electron microscopy (JEOL 2000EX operating at 100 kV) was used to characterize the morphology and structure of the gold nanoparticles. A 5 µL drop of the nanoparticle solution was placed onto a carbon-coated 200 mesh copper grid. Excess liquid was removed by contacting the side of the grid with absorbent paper tissue.
Results and Discussion Mannose-Stabilized Gold Nanoparticles. The development of an analytical colorimetric detection method based on metal nanoparticle aggregation demands that the initial optical signal be independent of the carbohydrate ligand used. The size of the nanoparticles formulated via reduction of the metal salt in the presence of the carbohydrate ligand is dependent upon both the amount and type of ligand. Such a method therefore would not be universally applicable as the particle size would be variable. For this reason, the mannose-stabilized gold nanoparticles were formulated by modification of preformed citrate-stabilized nanoparticles using displacement self-assembly with the thiolated mannopyranoside. The synthesis of the gold nanoparticles was achieved by citrate reduction of the gold salt to yield particles of 16 nm average diameter (as determined by TEM). The citrate reduction method ensured that the nanoparticles provide an intense surface plasmon band which can be used in the colorimetric assay. Upon modification with the thiolated mannose, the nanoparticles were isolated by multistep centrifugation and redispersed in Tris buffer at pH 7.6. In addition to isolating the mannose-stabilized gold nanoparticles, this procedure allows any unbound carbohydrate to be recovered from the supernatant. The UV-visible spectrum of the mannose-stabilized nanoparticles showed only a small 2 nm shift in the value of λMAX (from 521 to 523 nm, data not shown), indicating a change in the dielectric nature (26) Enu¨stu¨n, B. V.; Turkevich, J. J. Am. Chem. Soc. 1963, 85, 33173324.
Notes
Figure 1. TEM micrographs of mannose-stabilized gold nanoparticles (a) before and (b) 30 min after addition of 0.320 µM Con A. The overall concentration of nanoparticles in the sample remains the same in both (a) and (b).
of the metal surface due to the presence of the carbohydrate. Significantly, with consideration of future work in biological milieux, the mannose-stabilized nanoparticles are stable in buffered solution and in the presence of up to 4% v/v fetal calf serum as determined by UV-visible spectrophotometry. These results indicate that the modification of preformed gold nanoparticles with carbohydrates possessing short-chain thiol tethers provides a simple route to the formation of stable glyconanoparticles. Although experiments were undertaken using freshly prepared samples, no aggregation was observed within a buffered solution of the modified particles over a period of 2 months. Lectin-Induced Aggregation. At pH > 7, Con A exists as a tetramer27 and therefore would have four binding sites that are specific to R-D-mannose residues. At the pH used in this work (7.6), it is envisaged that the Con A tetramer would be able to bind up to four gold nanoparticles via the lectin-carbohydrate interaction. In addition, since there are multiple mannose ligands, each nanoparticle may simultaneously bind to more than one Con A tetramer. Consequently, when Con A was added to a solution of the mannose-stabilized gold nanoparticles significant aggregation occurred. Figure 1 shows the TEM micrographs obtained for the mannose-stabilized gold nanoparticles and the subsequent aggregation induced by the addition of Con A after a period of 30 min. Before addition of Con A, the particles are dispersed on the grid, and at the magnification shown, only 20 nanoparticles are observed (Figure 1a). On addition of Con A, aggregation occurs and the particles are brought together to form networks approaching micrometer length scales (Figure 1b). As the concentration of Con A increases, aggregation of the nanoparticles is enhanced. The surface plasmon absorption band shifts to longer wavelengths and shows (27) Senear, D. F.; Teller, D. C. Biochemistry 1981, 20, 3076-3083.
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Figure 2. Changes in the UV/visible spectra of mannosestabilized gold nanoparticles on addition of different concentrations of Con A: (a) 0, (b) 0.064 µM, (c) 0.128 µM, (d) 0.192 µM, (e) 0.256 µM, and (f) 0.320 µM. Spectra were obtained 40 min following addition of Con A.
a broadening which is consistent with aggregation of the particles, Figure 2. At all wavelengths between 600 and 800 nm, there is a quantitative concentration dependence on the measured absorbance. The data reported in Figure 2 were recorded 40 min following addition of Con A. It is thought that the particularly well-defined changes in the optical spectra, as a function of Con A concentration, are due to the short alkyl tether between the mannose and the gold surface. Mirkin et al.28 have shown that changes in the optical properties of DNA-linked systems are dependent upon the spacer length of the oligonucleotide and subsequent proximity of the nanoparticles in the aggregate. The molecular recognition between the mannose-stabilized nanoparticles and the Con A results in aggregates in which the nanoparticles are located in close proximity, separated only by the Con A tetramer (Figure 1b). To establish that the aggregation was induced by specific mannose recognition of the Con A, rather than electrostatic interactions, control experiments were carried out with a second lectin. Tetragonolobus purpureas has an affinity for L-fucose. When tetragonolobus purpureas was mixed with the mannose-stabilized nanoparticles, no change in the UV-visible absorption spectrum was observed, indicating that no interaction between the nanoparticles and the lectin occurred. It is possible to conclude, therefore, that the aggregation induced by Con A is specific to the mannose binding sites located around the particle surface and that such glyconanoparticles have potential use in the identification of unknown lectins. Aggregation Kinetics. From the data in Figure 2, it is clear that there is a significant change in absorbance at 620 nm which is dependent upon the concentration of Con A. Figure 3(i) shows the change in absorbance measured at 620 nm which occurs upon addition of Con A and the subsequent aggregation of the nanoparticles. At high concentrations of Con A (>0.3 µM), there is an initial sharp increase in absorbance which within 600 s slows, presumably as the recognition sites become occupied. To demonstrate that the mannose-stabilized nanoparticles can be used within a rapid assay format, the aggregation process over short time intervals has been studied. Figure 3(ii) again shows changes in the value of (28) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C. J. Am. Chem. Soc. 2001, 123, 8226-8230.
Figure 3. Change in the intensity of A620nm at varying concentrations of Con A, (a) 0.192 µM, (b) 0.256 µM, (c) 0.320 µM, and (d) 0.385 µM: (i) over 600 s; (ii) over 30 s; (iii) rate of change in A620nm versus Con A concentration.
A620nm with time, but importantly the response is over a 30 s period. As aggregation proceeds, there is the anticipated increase in absorbance which is dependent upon the concentration of Con A. Figure 3(iii) shows a plot of the rate of change in absorbance (slope of lines in Figure 3(ii)) versus concentration of Con A. The assay provides a linear response indicating that the mannosestabilized nanoparticles may be used to determine subµM Con A concentrations (0.192-0.385 µM) within 30 s. From the data in Figure 2, it is clear that lower concentrations (
