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Microarray-Based Study of Carbohydrate-Protein Binding by Gold Nanoparticle Probes Jingqing Gao,†,‡ Dianjun Liu,† and Zhenxin Wang*,† State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China In order to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate or glycoprotein microarrays by immobilizing amino modified carbohydrates on aldehydederivatized glass slides or glycoprotein on epoxidederivatized glass slides and carried out lectin binding experiments by using these microarrays, respectively. The interaction events are marked by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry. The detection principle is resonance light scattering (RLS). The well-defined recognition systems, namely, three monosaccharides (Man-r, Glc-β and Gal-β) or three glycoproteins (Asf, RNase A and RNase B) with two lectins (ConA and RCA120), were chosen here to establish the RLS assay, respectively. Highly selective recognition of carbohydrate-protein down to 25.6 pg/mL for RCA120 in solution and 8 µM for Gal-β and 32 ng/mL for Asf on the microarray spots is demonstrated. Cellular glycoconjugates play important roles in a wide variety of biological processes, including cell-cell communication, cell adhesion, fertilization, differentiation, development, inflammation, and tumor cell metastasis.1-7 Specific interactions between carbohydrates and proteins also initiate infection of host cells by bacteria and viruses since most host receptors or coreceptors of microbes are glycoconjugates.6,7 Therefore, understanding of the molecular basis for carbohydrate-protein interactions not only provides valuable information on biological processes in living organisms but also aids the development of potent biomedical agents.5-7 Many biophysical and biochemical methods have been * Author to whom correspondence should be addressed. E-mail: wangzx@ ciac.jl.cn. Fax:(+86) 431-5262243. † Changchun Institute of Applied Chemistry. ‡ Graduate School of the Chinese Academy of Sciences. (1) (a) Feizi, T. Adv. Exp. Med. Biol. 1982, 152, 167–177. (b) Feizi, T. Glycoconjugate J. 2000, 17, 553–565. (2) Rosati, F.; Capone, A.; Giovampaola, C. D.; Brettoni, C.; Focarelli, R. Int. J. Dev. Biol. 2000, 44, 609–618. (3) Wang, D.; Liu, S.; Trummer, B. J.; Deng, C.; Wang, A. Nat. Biotechnol. 2002, 20, 275–281. (4) Danica, P. G.; David, Y. G. Nature 2007, 446, 1000–1007. (5) Varki, A. Glycobiology 1993, 3, 97–130. (6) Sharon, N.; Lis, H. Glycobiology 2004, 14, 53R–62R. (7) Peters, T.; Scheffler, K.; Ernst, B.; Katopodis, A.; Magnani, J. L.; Wang, W. T.; Weisemann, R. Angew. Chem., Int. Ed. Engl. 1995, 34, 1841–1844.
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used to study the details of carbohydrate-protein interactions.8-12 For example, X-ray crystallographic and NMR spectroscopic techniques have been employed to determine binding modes between carbohydrates and proteins.8,9 Surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) have been used to provide information on the binding affinities of carbohydrates to proteins.10,11 And specifically modified synthetic carbohydrates have been used to elucidate the molecular basis of carbohydrate-protein interactions.12 Recently, more and more attention has been given to development of microarray-based high-throughput methods to probe the carbohydrate-protein interactions because DNA microarrays and protein microarrays have been successfully fabricated and utilized in genomic, transcriptomic, and proteomic investigations.4,13-23 Currently, these microarray techniques mainly rely on the use of fluorescent molecular dye labels which have several potential (8) Somers, W. S.; Tang, J.; Shaw, G. D.; Camphausen, R. T. Cell 2000, 103, 467–479. (9) (a) Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387–430. (b) Green, R. J.; Frazier, R. A.; Shakesheff, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 2000, 21, 1823–1835. (c) Mann, D. A.; Kanai, M.; Maly, D. J.; Kiessling, L. L. J. Am. Chem. Soc. 1998, 120, 10575–10582. (10) (a) Ratner, D. M.; Adams, E. W.; Su, J.; O’Keefe, B. R.; Mrksich, M.; Seeberger, P. H. ChemBioChem 2004, 5, 379–383. (b) Karamanska, R.; Clarke, J.; Blixt, O.; MacRae, J. I.; Zhang, J. Q.; Crocker, P. R.; Laurent, N.; Wright, A.; Flitsch, S. L.; Russell, D. A.; Field, R. A. Glycoconj. J. 2007, 69–74. (c) Wakao, M.; Saito, A.; Ohishi, K.; Kishimoto, Y.; Nishimura, T.; Sobel, M.; Suda, Y. Bioorg. Med. Chem. Lett. 2008, 18, 2499–2504. (11) (a) Perou, C. M. Nature 2000, 406, 747–752. (b) Ramsey, G. Nat. Biotechnol. 1998, 16, 40–44. (c) Marshall, A.; Hodgson, J. Nat. Biotechnol. 1998, 16, 27–31. (d) DeRisi, J. L.; Lyer, V. R.; Brown, P. O. Science 1997, 278, 680–686. (e) Filser, C.; Kowalczyk, D.; Jones, C.; Wild, M. K.; Ipe, U.; Vestweber, D.; Kunz, H. Angew. Chem., Int. Ed. 2007, 46, 2108–2111. (12) Blixt, O.; Han, S.; Liao, L.; Zeng, Y.; Hoffmann, J.; Futakawa, S.; Paulson, J. C. J. Am. Chem. Soc. 2008, 130, 6680–6681. (13) Huang, C. Y.; Thayer, D. A.; Chang, A. Y.; Best, M. D.; Hoffmann, J.; Head, S.; Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15–20. (14) Nimrichter, L.; Gargir, A.; Gortler, M.; Altstock, R. T.; Shtevi, A.; Weisshaus, O.; Fire, E.; Dotan, N.; Schnaar, R. L. Glycobiology 2004, 14, 197–203. (15) Disney, M. D.; Seeberger, P. H. Chem. Biol. 2004, 11, 1701–1707. (16) de Paz, J. L.; Seeberger, P. H. QSAR Comb. Sci. 2006, 25, 1027–1032. (17) Park, S.; Lee, M. R.; Pyo, S. J.; Shin, I. J. Am. Chem. Soc. 2004, 126, 4812– 4819. (18) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443–454. (19) Zhou, X. C.; Zhou, J. H. Biosens. Bioelectron. 2006, 21, 1451–1458. (20) Manimala, J. C.; Roach, T. A.; Li, Z. T.; Gildersleeve, J. C. Angew. Chem., Int. Ed. 2006, 45, 3607–3610. (21) Mellet, C. O.; Garcia Fernandez, J. M. ChemBioChem 2002, 3, 819–822. (22) Chevolot, Y.; Bouillon, C.; Vidal, S.; Morvan, F.; Meyer, A.; Cloarec, J. P.; Jochum, A.; Praly, J. P.; Vasseur, J. J.; Souteyrand, E. Angew. Chem., Int. Ed. 2007, 46, 2398–2402. (23) Panicker, R. C.; Chattopadhaya, S.; Yao, S. Q. Anal. Chim. Acta 2006, 556, 69–79. 10.1021/ac8015328 CCC: $40.75 2008 American Chemical Society Published on Web 10/15/2008
drawbacks, such as the lack of sensitivity and photoinstability of the dyes employed.24,25 The metal, semiconductor, and magnetic nanoparticles can offer a unique set of physical properties that may be exploited in biological detection assays as an alternative to the use of fluorescent dyes.26 Particularly attractive is the use of gold and silver nanoparticles in resonant light scattering (RLS), which represents a great step forward, toward higher sensitivity, with the eventual goal of detecting single biomolecular binding events.25,26a,b As a pioneer, Mirkin and co-workers have developed a DNA modified gold nanoparticle-based assays for highly sensitive and selective detection of DNAs and proteins on microarrays.25,27 In these assays, DNA-gold nanoparticle conjugates are used as labels to detect specific analytes, i.e., DNA or proteins, followed by a RLS enhancement step based on the electroless deposition of silver onto the gold particles. Using same detection principle, a microarray format for detection of proteins and protein functionality (kinase activity) by gold nanoparticle probes has also been developed by Wang et al.28 In this paper, we developed a microarray based RLS assay for detection of carbohydrate-lectin or glycoprotein-lectin interactions by attachment of gold nanoparticles followed by silver deposition for signal enhancement. In the proof-of-concept experiments, we demonstrate that it is possible to (i) identify carbohydrate-lectin (ManR-ConA and Gal-β-RCA120) or glycoprotein-lectin (Asf-RCA120 and RNase B-ConA) interactions, and (ii) obtain a sensitive and selective assay for detection carbohydrates, lectins and glycoproteins. EXPERIMENTAL SECTION Materials and Reagents. Amino modified monosaccharides (4-aminophenyl R-D-mannopyranoside (Man-R), 4-aminophenyl β-D-galactopyranoside (Gal-β) and 4-aminophenyl β-D-glucopyranoside (Glc-β)), glycoproteins (ribonuclease B (RNase B), ribonuclease A (RNase A) and asialofetuin (Asf)), biotinylated concanavalin A from Canavalia ensiformis (ConA-biotin), fluorescein isothiocyanate labeled avidin (avidin-FITC), bovine serum albumin (BSA), tetrachloroaurate (HAuCl4) and silver enhancer and were obtained from Sigma Corp. (USA) and using as received. Biotinylated Ricinus communis agglutinin (RCA 120-biotin) was obtained from Vector Laboratory Ltd. (Burlingame, CA). CALNN was purchased from Sangon Ltd. (Shanghai, China). CALNNGK(biotin)G was purchased from Scilight Biotechnology Ltd. (Beijing, China). Aldehyde or epoxide modified glass microscope slides were obtained from CapitalBio (Beijing, China). Other chemicals (24) (a) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119. (b) Kodadek, T. Chem. Boil. 2001, 8, 105– 115. (c) Schena, M. Microarray analysis; Wiley-Liss: Hoboken, NJ, 2003. (25) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760. (26) (a) Yguerabide, J.; Yguerabide, E. E. J. Cell. Biochem. 2001, 37 (suppl.), 71–81. (b) Oldenburg, S. J.; Genick, C. C.; Clark, K. A.; Schultz, D. A. Anal. Biochem. 2002, 309, 109–116. (c) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (d) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47–52. (e) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042– 6108. (f) You, C.-C.; De, M.; Rotello, V. M. Curr. Opin. Chem. Biol. 2005, 9, 639–646. (g) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. Anal. Chem. 2008, 80, 1067–1072. (27) (a) Cao, Y. C.; Jin, R.; Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676–14677. (b) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886. (28) (a) Wang, Z. X.; Lee, J.; Cossins, A. R.; Brust, M. Anal. Chem. 2005, 77, 5770–5774. (b) Sun, L. L.; Liu, D. J.; Wang, Z. X. Anal. Chem. 2007, 79, 773–777.
were analytical grade and used as received. Milli-Q water (18.2 MΩ) was used in all experiments. Preparation of Gold Nanoparticles and Peptide-Stabilized Nanoparticles. Thirteen nanometer gold nanoparticles were synthesized by the traditional “Turkevich-Frens” method.29 Peptide-stabilized nanoparticles were prepared by previously reported peptide capping procedure.30 Generally, an aqueous solution of peptide mixture (CALNN: CALNNGK(biotin)G) was added to the solution of 5 nM 13 nm gold nanoparticles to give a final concentration of total peptide of 1.5 mM. The ratio of CALNN and CALNNGK(biotin)G in the mixture is 9:1. After 1 h incubation, excess peptides were removed by repeated centrifugation at 13000 rpm (∼16100g, 3×) using an Eppendorf centrifuge (Eppendorf, Germany). The purified gold nanoparticles were resuspended in 50 mM PBS and then stored at 4 °C. Carbohydrate Microarrays. Aldehyde modified glass microscope slides were used to fabricate carbohydrate microarrays by the standard procedure with a SmartArrayer 48 system (Capitalbio Ltd., China).31 The aldehyde groups on the glass surface react readily with the primary amines of the amino modified monosaccharides to form a Schiff base linkage. Monosaccharides with desired concentration were dissolved in 15 µL of spotting buffer (pH 8.5, 0.3 M PB, 0.15 M NaCl supplemented with 0.005% (v/v) Tween-20) with 15% (v/v) glycerol and spotted on the glass microscope slides by contact printing. After an overnight incubation under 75% humidity at 25 °C, the slides were rinsed with 50 mL of washing buffer (pH 7.5, 50 mM, PB supplemented with 0.05% (v/v) Tween-20; 3 times) and then immersed in 10 mL of blocking buffer (pH 7.5, 50 mM PB, 0.15 M NaCl supplemented with 1% (w/w) BSA and 0.1 M ethanolamine) at 30 °C for 1 h to remove remaining free aldehyde groups. Each array was incubated with biotin-modified lectins, ConA and RCA 120, which were diluted to the desired concentration with 20 µL of probe buffer (50 mM PB, 0.15 M NaCl, 1 mM CaCl2, 1 mM MnCl2 and 1% BSA (w/w)). Following 1 h incubation at 25 °C, the slides were subjected to a series of rinsing steps: (1) 50 mL of probe buffer for 3 min (3 times); (2) 50 mL of washing buffer for 3 min (3 times); (3) 50 mL of PBS buffer for 3 min (3 times) and (4) 50 mL of Milli-Q water for 3 min (18.2 MΩ, 3 times), then dried by centrifugation (480 g for 1 min). Then array was incubated with 20 µL avidin-FITC solution (50 µg/mL avidin-FITC in 50 mM PB containing 0.15 M NaCl and 1% BSA (w/w)) washed with washing buffer, PBS buffer and water as described before, then dried by centrifugation. Subsequently, the array was treated with 20 µL of a solution of peptide-stabilized gold nanoparticles (2.5 nM) in probe buffer for 1 h at 37 °C, then washed and dried as described before. Glycoprotein Microarrays. Glycoproteins were spotted on epoxide functionalized slides in PBS spotting buffer with 40% glycerol (v/v) included to prevent evaporation of the nanodroplets. In here, the primary amines on the glycoprotein surface act as nucleophiles, attacking epoxy groups and coupling the protein (29) (a) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55–75. (b) Frens, G. Nat. Phys. Sci. 1973, 241, 20–22. (30) (a) Levy, R.; Nguyen, T. K.; Thanh, R.; Doty, C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076–10084. (b) Wang, Z. X.; Levy, R.; Fernig, D. G.; Brust, M. Bioconjugate Chem. 2005, 16, 497–500. (31) (a) http://cmgm.stanford.edu/pbrown/. ((b)) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2, research 0004.1-0004.13.
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Figure 1. Schematic representation of one spot on the microarray showing, from left to right, the binding of a biotin-modification lectin to the spotted monosaccharide (a) and glycoprotein (b), then labeled by avidin-FITC followed by the attachment of peptide stabilized gold nanoparticles, the silver enhancement step and reading by detection of resonant light scattering. For comparison, the microarray can also be detected by a conventional fluorescence microarray scanner before treatment with the gold nanoparticles.
covalently to the surface. After an overnight reaction at 25 °C in a vacuum environment, the slides were washed and blocked as described in the carbohydrate microarrays fabrication. After blocking, each array was incubated with biotinylated lectins, ConAbiotin and RCA 120-biotin and washed, dried as previously described. Then array was subjected to label with 20 µL avidinFITC solution (50 µg/mL avidin-FITC in 50 mM PB containing 0.15 M NaCl and 1% BSA (w/w)) and treated with 20 µL of 2.5 nM peptide-stabilized gold nanoparticles as described in the Carbohydrate Microarrays section, respectively. Silver Enhancement and Detection. After being treated with gold nanoparticles, silver enhancer (1:1 mixed (1 mL total volume) solutions A (AgNO3) and B (hydroquinone) (Sigma-Aldrich)) was applied to each microarray for several minutes and washed with water (3 times). As previously reported, in this study all results shown were obtained under optimum conditions (8 min exposure time) since the detection sensitivity and dynamic range was critically related to the amount of silver deposited.28 After signal amplification by silver deposition, the slides were detected with ArrayIt SpotWare Colorimetric Microarray Scanner (TeleChem. International Inc.). According to the manufacturer’s preset parameters, all images were collected with broad spectrum white light source.32 The background originating from the slide was recorded and subtracted from each image prior to evaluation. The mean value and standard deviation of the signal were determined for the 25 or 36 spot replicates per sample, respectively. The detection limit was determined to be the concentration where signal/standard deviation ) 3 was reached. For the determination of the linear ranges of the curves, the range of concentrations that best fitted the linear equation y = mx + b were specified. (32) http://www.arrayit.com/Products/MicroarrayI/Scanner/scanner.html.
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RESULTS AND DISCUSSION Carbohydrate Microarrays. Monosaccharides were spotted and immobilized on commercial aldehyde-functionalized glass microscope slides by a standard robotic procedure.31 The binding scheme and detection rationale employed is schematically shown in Figure 1a. For comparison, we design a microarray format for detection of carbohydrate-protein binding with two detection principles: conventional fluorescence detection and RLS detection. After recognition of the immobilized monosaccharides by biotinylated lectins, avidin-conjugated fluorescein isothiocyanate (avidin-FITC) is used to label the recognition event. At this step, the microarrays are ready for fluorescence detection. Here, the fluorescein plays as a fluorescence-active probe which can be detected by conventional fluorescence microarray scanner and the avidin is used as a linker for attaching gold nanoparticles to the system for subsequent RLS detection. Then, peptide-stabilized gold nanoparticles are attached to these recognition events by avidin-biotin reaction. Subsequently, a silver enhancement step is applied to the microarrays for signal amplification since the light scattering properties of gold nanoparticles by themselves are relatively poor, if the particles are smaller than ca. 40 nm.25,26 After treatment with the silver enhancement solution, the microarrays are readily detected by the RLS microarray scanner. The structural detail of the particles after silver deposition is shown in Figure S1 in the Supporting Information. To detect specific monosaccharide-lectin interactions on the microarray, three different monosaccharides, Man-R, Glc-β and Gal-β, were spotted on the slide to be probed with two different lectins (ConA and RCA120), which were chosen for their wellknown specificities.33 RCA120 binds only to Gal-β, while ConA binds to both Man-R and Glc-β. In addition, the affinity of ConA with Man-R is stronger than that of ConA with Glc-β.17,19 All lectins
Figure 2. Light scattering images and corresponding analysis of monosaccharide microarrays after probing with lectins, labeling positives with gold nanoparticles and enhancement by silver deposition. Man-R was spotted on column 1, Glc-β was spotted on column 2, and Gal-β was spotted on column 3. Each column contains 5 replicate spots. The microarrays were probed with ConA (a) and RCA120 (b). The concentration of spotted monosaccharide is 50 mM and probe lectin is 50 µg/mL, respectively.
Figure 3. Images of microarrays and corresponding logarithmic plots of the integrated light scattering intensity as a function of the concentrations of Gal-β in the spotting solution (a) and RCA120 in the probe solution (b). Each column contains 6 replicate spots. In (a) the concentration of the RCA120 in the probe solution was 1.5 µg/mL, and in (b) the concentration of Gal-β was 25 mM in spotting solution. Table 1. The Detection Limits and Dynamic Ranges of the RLS or Fluorescence Assay with Monosaccharide Microarray Format RLS assay
fluoresence assay
analytes
detection limit
dynamic range
detection limit
dynamic range
Man-Ra ConAb Gal-βa RCA120b
100 µM 100 pg/mL 8 µM 100 pg/mL
0.5-50 mM 0.5-500 ng/mL 0.2-100 mM 400 pg/mL to 1.5 µg/mL
500 µM 1 µg/mL 200 µM 300 ng/mL
0.5 -50 mM 1-50 µg/mL 1-50 mM 0.3-100 µg/mL
a The detection limit and dynamic range of monosaccharide (Man-R or Gal-β) are based on the initial concentration of spotting solution, and the concentration of the lectin (RCA120 or ConA) in the probe solution is 1.5 µg/mL. b The concentration of monosaccharide (Man-R or Gal-β) is 25 mM in spotted solution.
used are modified with biotin. The results of this array-based binding assay are shown in Figure 2 (corresponding fluorescent experimental results are shown in Figure S2 in the Supporting Information). As anticipated, only the spots probed with specifically binding lectin give a positive signal, i.e. RCA120 only recognizes Gal-β, and ConA, as expected, recognizes both Man-R and Glc-β. In particular, the binding affinities of ConA with Man-R or Glc-β can be clearly discriminated from the RLS intensity. These results demonstrate that the immobilized monosaccharides retain (33) (a) Lee, Y. C.; Lee, R. T.; Rice, K.; Ichikawa, Y.; Wong, T. C. Pure Appl. Chem. 1991, 63, 499–506. (b) Sharon, N.; Lis, H. Essays Biochem. 1995, 30, 59–75. (c) Elgavish, S.; Shaanan, B. Trends Biochem. Sci. 1997, 22, 462–467.
their recognition function on the glass surface and, more importantly, that any nonspecific binding of lectin is below the detection limit. Quantitative Detection. A set of experiments was designed to determine the detection limits of the method described. By varying the concentration of Gal-β in the spotting solution while keeping the RCA120 concentration of the probing solution constant the optimal signal-to-noise ratio (three times of the standard deviation) is obtained at spotting solution concentrations above 8 µM indicating the detection limit is 8 µM for Gal-β which is less than the typical concentration of the carbohydrate (ca. 10 mM) in the biological system (e.g. plants).34 This signal increased (34) Lee, D. H.; Kang, S. G.; Suh, S. G.; Byun, J. K. Mol. Cells 2003, 15, 68–74.
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Figure 4. Light scattering images and corresponding analysis of glycoprotein microarrays after probing with lectins, labeling positives with gold nanoparticles and enhancement by silver deposition. RNase B was spotted on column 1, Asf was spotted on column 2, RNase A was spotted on column 3 and BSA was spotted on column 4. Each column contains 6 replicate spots. The microarrays were probed with ConA (a) and RCA120 (b). The concentration of spotted glycoprotein is 0.5 mg/mL and probe lectin is 50 µg/mL, respectively.
Figure 5. Images of microarrays and corresponding logarithmic plots of the integrated light scattering intensity as a function of the concentrations of Asf in the spotting solution (a) and RCA120 in the probe solution (b). Each column contains 6 replicate spots. In (a) the concentration of the RCA120 in the probe solution was 2 µg/mL and in (b) the concentration of Asf was 250 µg/mL in the spotting solution.
linearly with the logarithm of the Gal-β concentration from 0.2 to 100 mM indicating a dynamic range of nearly 3 orders of magnitude as shown in Figure 3a. For the lectin in solution much lower concentrations are needed. For example, specific binding could be detected using RCA 120 at concentrations as low as 100 pg/mL as shown in Figure 3b. And this signal increased linearly with the logarithm of the RCA120 concentration from 400 pg/mL to 1.5 µg/mL indicating a dynamic range of 3 orders of magnitude. On the other hand, the conventional fluorescence techniques for detecting the same target (RCA120) on same microarray have a detection limit of 300 ng/mL, i.e. 3 orders of magnitude less sensitive (as shown in Figure S3 in the Supporting Information). Corresponding experimental results of the interaction of Man-R with ConA are shown in Figure S4 in the Supporting Information. For comparison, the detection limits and dynamic ranges of the assays are summarized in Table 1. Generally, the detection limit and dynamic range of the RLS assay are better than those of fluorescent assay. These results indicate that the prospects for clinical application of our approach in monitoring lectins response to immunization is extremely promising since a natural immune response typically yields specific lectin concentrations over 2 ng/ mL.35 Glycoprotein Microarrays. The reaction scheme for the determination of glycoprotein-lectin interaction is shown in Figure 1b. The immobilized glycoproteins are recognized, labeled and treated as described for carbohydrate microarray. Three (35) Kilpatrick, D. C. Transfusion Med. 2002, 12, 335–351.
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glycoproteins, RNase B, RNase A and Asf, are spotted and immobilized on epoxide functionalized slides. Selective binding of the three glycoproteins with two lectins, ConA and RCA 120, are studied. In this case, RCA120 binds Asf, while ConA binds to RNase B.36 Figure 4 shows the selective interaction of glycoproteins with lectins. This demonstrates the high selectivity of this detection method, i.e. the absence of detectable nonspecific attachment of gold nanoparticles. The experimental result is consistent with the fluorescent study (see Figure S5 in the Supporting Information). Quantitative Detection. The sensitivity of this method was examined by varying the concentrations of glycoproteins in the spotting solution while keeping the concentrations of lectins constant or keeping the concentrations of glycoproteins constant in the spotting solution while varying the concentrations of lectins. The results of the interaction of Asf with RCA120 are shown in Figure 5. An optimal signal-to-noise ratio was obtained at concentrations of Asf above 32 ng/mL while keeping RCA120 at 2 µg/ mL or concentrations of RCA120 above 25 pg/mL while keeping Asf at 250 µg/mL. At this detection limit (32 ng/mL) the actual total amount of Asf on each spot (1 nL spotting volume) is equal to or less than 32 fg. For further comparison, the corresponding fluorescent studies are shown in Figures S6 and S7 in the Supporting Information, and the detection limits and dynamic (36) Kuno, A.; Uchiyama, N.; Koseki-Kuno, S.; Ebe, Y.; Takashima, S.; Yamada, M.; Hirabayashi, J. Nat. Methods 2005, 2, 851–856.
Table 2. The Detection Limits and Dynamic Ranges of the RLS or Fluorescence Assay with Glycoprotein Microarray Format RLS assay
fluoresence assay
analytes
detection limit
dynamic range
detection limit
dynamic range
Asfa RCA120b RNase Ba ConAb
32 ng/mL 25.6 pg/mL 0.16 µg/mL 0.5 ng/mL
0.16-500 µg/m l 0.003-10 µg/mL 0.8-1000 µg/mL 1-1000 ng/mL
4 µg/mL 400 ng/mL 0.8 µg/mL 100 ng/mL
0.02-1 mg/mL 2-100 µg/mL 4-500 µg/mL 0.5-10 µg/mL
a The detection limit and dynamic range of glycoprotein (Asf or RNase B) are based on the initial concentration of spotting solution, and the concentration of the lectin (RCA120 or ConA) in the probe solution is 2 µg/mL. b The concentration of glycoprotein (Asf or RNase B) is 250 µg/mL in spotted solution.
ranges are summarized in Table 2. These results indicate that our method could be used to detect glycoproteins in various body fluids which typically yields specific glycoprotein (e.g., RNase B) concentrations ranging from approximately 0.1 µg/mL to over 1 µg/mL.37
tion or characterization of novel carbohydrate-binding proteins in cell or tissue extracts and discovery of new roles for carbohydrates in cell biology, and it shows great promise for large-scale glycoproteome screening. ACKNOWLEDGMENT The authors thank the NSFC (Grant No. 20675080, 20875087) for financial support.
CONCLUSIONS A versatile gold nanoparticle-based carbohydrate or glycoprotein microarray format with high selectivity and sensitivity for carbohydrate-protein binding has been developed. Compared to fluorescence readout format, our RLS approach has reasonably lower detection limits for carbohydrate-protein binding combined with a larger dynamic range. Well-known biomolecular recognition systems were chosen here to establish this new microarray format by proof-of-principle experiments. However, our approach is readily transferable to real analytical problems such as identifica-
SUPPORTING INFORMATION AVAILABLE RLS images of the interactions of Man-R or RNase B with ConA; fluorescent images of the monosaccharides and glycoproteins microarrays. This material is available free of charge via the Internet at http://pubs.acs.org.
(37) Levy, A. L.; Rottino, A. Clin. Chem. 1960, 6, 43–51.
AC8015328
Received for review July 22, 2008. Accepted September 26, 2008.
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