Detection of Carbohydrate Binding Proteins Using Magnetic

Aug 4, 2010 - Address: Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio, 45221-0172. Phone: (513) 556-9273. Fax: (...
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Anal. Chem. 2010, 82, 7430–7435

Detection of Carbohydrate Binding Proteins Using Magnetic Relaxation Switches Ashish A. Kulkarni,† Alison A. Weiss,‡ and Suri S. Iyer*,† Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, and Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0524 We have developed a simple, rapid, and sensitive carbohydrate-based magnetic relaxation switch assay for the detection of carbohydrate binding proteins. This technique was used to detect lectins and toxins that are known to bind to specific carbohydrates. Lectins that bind to the same carbohydrate displayed differential aggregation profiles because of differences in the structure and number of binding sites of the lectins. We demonstrated that selectivity and sensitivity can be enhanced using two different recognition elements. We have also demonstrated that magnetic relaxation switch assays can be used to detect toxins in a complex medium such as stool and environmental samples. Nature’s third class of biopolymers, carbohydrates, are excellent recognition molecules that could be harnessed for a variety of biotechnological applications that include targeted drug/gene delivery, therapeutics, and diagnostics.1,2 In addition to their excellent recognition capability, carbohydrates are highly robust, inexpensive, and require no refrigeration and manufacture can be scaled up readily, qualities that make them uniquely suitable as recognition elements of biosensors.3,4 Carbohydrate based biosensors are particularly useful to detect antigenic variants because carbohydrate based recognition is a receptor binding strategy that cannot be altered without loss of virulence.3,5 Additionally, the smaller size of carbohydrates provides them with a unique size advantage over larger recognition elements such as antibodies or aptamers, and therefore, carbohydrates could find use for potential applications in nanobiosensors, where size of the recognition element is a key requirement. Several reports, including our own work, have reported several approaches to harness the recognition potential of carbohydrates for biosensors. Carbohydrates attached to a variety of platforms * To whom correspondence should be addressed. Address: Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio, 452210172. Phone: (513) 556-9273. Fax: (513) 556-9239. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Molecular Genetics, Biochemistry, and Microbiology. (1) Bertozzi, C. R.; Freeze, H. H.; Varki, A.; Esko, J. D. In Essentials of Glycobiology, 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor Laboratory Press Plainview: New York, 2008; pp 719-732. (2) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046–1051. (3) Hatch, D. M.; Weiss, A. A.; Kale, R. R.; Iyer, S. S. ChemBioChem 2008, 2433–2442. (4) Disney, M. D.; Seeberger, P. H. Chem. Biol. 2004, 11, 1701–1707. (5) Kale, R. R.; McGannon, C. M.; Fuller-Schaefer, C.; Hatch, D. M.; Flagler, M. J.; Gamage, S. D.; Weiss, A. A.; Iyer, S. S. Angew. Chem., Int. Ed. Engl. 2008, 47, 1265–1268.

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that include polymers,6 noble metal,7 magnetic,8,9 fluorescent nanoparticles,10 and dendrimers11 have been used to detect lectins,12,13 toxins,14 bacteria,15,16 viruses,17 and cancerous cells.18 Recently, we compared the recognition capabilities of carbohydrate coated micrometer magnetic beads to antibody coated magnetic beads.3 In this study, we report a one step, no wash, magnetic relaxation assay that used carbohydrate coated micrometer magnetic beads to detect carbohydrate binding proteins with high sensitivity and minimal sample preparation. After optimizing conditions and developing this bioassay using model lectins, we used it to detect two different isoforms of Shiga toxins. The bioassay provides quantitative concentration data within 1 h with an observed detection limit of 1 pg/mL of these toxins in complex media such as stool samples. This simple “add and measure” assay could potentially be used by first responders to detect infectious agents. EXPERIMENTAL SECTION We describe briefly the experimental procedures for the synthesis of the carbohydrates, procedures for the attachment of the carbohydrate to the magnetic micrometer beads, complete characterization of the carbohydrate coated magnetic micrometer beads using multiple techniques, and measurement of the transverse relaxation time under various conditions. Part of the (6) Disney, M. D.; Zheng, J.; Swager, T. M.; Seeberger, P. H. J. Am. Chem. Soc. 2004, 126, 13343–13346. (7) de la Fuente, J. M.; Penades, S. Biochim. Biophys. Acta 2006, 1760, 636– 851. (8) El-Boubbou, K.; Gruden, C.; Huang, X. J. Am. Chem. Soc. 2007, 129, 13392– 13393. (9) van Kasteren, S. I.; Campbell, S. J.; Serres, S.; Anthony, D. C.; Sibson, N. R.; Davis, B. G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18–23. (10) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H. J. Am. Chem. Soc. 2009, 131, 2110–2112. (11) Kikkeri, R.; Hossain, L. H.; Seeberger, P. H. Chem. Commun. (Cambridge, U.K.) 2008, 2127–2129. (12) Schofield, C. L.; Mukhopadhyay, B.; Hardy, S. M.; McDonnell, M. B.; Field, R. A.; Russell, D. A. Analyst 2008, 133, 626–634. (13) Lin, C. C.; Yeh, Y. C.; Yang, C. Y.; Chen, G. F.; Chen, Y. C.; Wu, Y. C.; Chen, C. C. Chem. Commun. (Cambridge, U.K.) 2003, 2920–2921. (14) Schofield, C. L.; Field, R. A.; Russell, D. A. Anal. Chem. 2007, 79, 1356– 1361. (15) 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. (16) Xue, C.; Velayudham, S.; Johnson, S.; Saha, R.; Smith, A.; Brewer, W.; Murthy, P.; Bagley, S. T.; Liu, H. Chemistry 2009, 15, 2289–2295. (17) Kale, R. R.; Mukundan, H.; Price, D. N.; Harris, J. F.; Lewallen, D. M.; Swanson, B. I.; Schmidt, J. G.; Iyer, S. S. J. Am. Chem. Soc. 2008, 130, 8169–8171. (18) El-Boubbou, K.; Zhu, D. C.; Vasileiou, C.; Borhan, B.; Prosperi, D.; Li, W.; Huang, X. J. Am. Chem. Soc. 2010, 132, 4490–4499. 10.1021/ac101579m  2010 American Chemical Society Published on Web 08/04/2010

experimental procedures can be found in the Supporting Information section. The major abbreviations used throughout the paper are GC1: biotinylated tetra-antennary R-mannoside; GC2: biotinylated monoantennary R-mannoside; GC3: biotinylated monoantennary β-galactoside, Pk: biotinylated biantennary trisaccharide specific for Shiga toxin 1; NHAc-Pk: biotinylated biantennary trisaccharide specific for Shiga toxin 2; GNL: Galanthus Nivalis lectin; RCA120: Ricinus communis lectin; ConA: Concanavalin A; Stx1: Shiga toxin 1; Stx2: Shiga toxin 2. General Methods for the Synthesis of the Tailored Carbohydrates. All chemical reagents were of analytical grade, used as supplied without further purification unless indicated. Acetic anhydride and acetyl chloride were distilled under an inert atmosphere and stored under argon. Molecular sieves (4 Å) were stored in an oven (>130 °C) and cooled in vacuo. The acidic ionexchange resin used was Dowex-50 and Amberlite (H+ form). Analytical thin layer chromatography (TLC) was conducted on silica gel 60-F254 (Merck). Plates were visualized under UV light and/or by treatment with acidic cerium ammonium molybdate followed by heating. Column chromatography was conducted using silica gel (230-400 mesh) from Qualigens. 1 H and 13C NMR spectra were recorded on Bruker AMX 400 MHz spectrometer. Chemical shifts are reported in δ (ppm) units using 13C and residual 1H signals from deuterated solvents as references. Spectra were analyzed with Mest-Re-C Lite (Mestrelab Research) and/or XWinPlot (Bruker Biospin). Electrospray ionization mass spectra were recorded on a Micromass Q Tof 2 (Waters), and data were analyzed with MassLynx 4.0 (Waters) software. Synthesis of Mannose Based Tetra-Antennary Carbohydrates. Compound 6: 1-O-(6-t-Butylcarbamate-4-thiohexyl)-R-Dgalactopyranoside. 1-O-(6-t-Butylcarbamate-4-thiohexyl)-2,3,4,6-tetraO-acetyl-R-D-galactopyranoside19 (0.98 g, 2.5 mmol) was dissolved in MeOH (20 mL) and cooled to 0 °C. A solution of NaOMe in MeOH (0.5M, 2.5 mL) was added via syringe, and the resulting mass was stirred at room temperature (RT) for 6 h. Upon completion (TLC), the reaction was neutralized by careful addition on Amberlite-15 H+ resin and filtered. The solvent was removed in vacuo, and the residue was purified by flash column chromatography, eluting with an 80:20 mixture of CH2Cl2 and methanol system to give 6 as white solid (0.61 g, 89%). Spectral data matched reported values.20 Compound 7: 1-O-(6-t-Butylcarbamate-4-thiohexyl)-2,3,4,6-tetraO-propargyl-R-D-galactopyranoside. NaH (60% in oil, 0.61 g, 6.93 mmol) was washed with hexane. Compound 6 (0.61 g, 1.54 mmol) was dissolved in tetrahydrofuran (THF; 25 mL) and added to it. Next, propargyl bromide (0.60 g, 6.93 mol) was added via syringe, and the mixture was refluxed under an argon atmosphere for 1 h. Upon completion (TLC), the mass was cooled to 0 °C and carefully quenched with ethanol. The reaction mixture was diluted with 30 mL of water, and the organic layer was separated. The aqueous fraction was washed with 2 × 100 mL of EtOAc. The combined organic layer was dried over Na2SO4 and filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography, eluting with 50:50 mixture of EtOAc and hexane, to give 7 as a white solid (0.43 g, 51%). 1H NMR

(CDCl3): δ 5.03 (d, J ) 3.6 Hz, 1H), 4.49 (m, 2H), 4.4-4.1 (m, 6H), 3.98-3.95 (m, 2H), 3.90 (dd, J ) 2.8 Hz, J ) 10 Hz, 1H), 3.813-3.752 (m, 2H), 3.67-3.55 (m, 2H), 3.32-3.30 (m, 2H), 2.66-2.61 (m, 4H), 1.94-1.87 (m, 2H), 1.44 (bs, 9H). 13C NMR (CDCl3): δ 155.77, 97.52, 80.24, 80.13, 80.07, 79.54, 78.34, 76.06, 74.77, 74.72, 74.69, 74.59, 74.49, 69.09, 69.04, 66.43, 59.76, 58.81, 58.72, 58.65, 39.63, 32.20, 29.31, 28.42, 28.33, 28.29, 28.24. HRMS calculated for [C28H39NO8SNa]+, 572.2294; found, 572.2314. Compound 10. Compound 7 (0.20 g, 0.36 mmol) in 1:1 THF and water was added to compound 821 (0.74 g, 1.60 mmol) via canula. Sodium acrobate (0.29 g, 1.45 mmol) and CuSO4 (0.18 g, 0.73 mmol) were added to it and stirred at RT for 48 h. Upon completion (TLC), solvent was removed in vacuo. The crude product was dissolved in EtOAc and washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to get 9. This crude product was used without further purification for subsequent steps.The crude product was dissolved in anhydrous CH2Cl2 (6 mL), and TIPS (50 µL) was added to it via syringe. The reaction mixture was cooled to 0 °C. TFA (350 µL) was added dropwise and stirred at RT for 24 h. Upon completion (TLC), the solvent was removed in vacuo. The residue was diluted with CH2Cl2 and washed with saturated NaHCO3. The organic layer was separated and dried over anhydrous Na2SO4 and filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography, eluting with a 94:6 mixture of CH2Cl2 and MeOH to give 10 as a light yellow solid (0.42 mg, 50%). 1H NMR (CDCl3): δ 7.89-7.74 (m, 4H), 5.31-5.24 (m,12H), 4.973-4.735 (m, 10H), 4.553 (bs, 10H), 4.299-4.258 (m, 4H), 4.1-3.5 (m, 40H), 3.22 (s, 1H), 2.157 (s, 12H), 2.094 (s, 12H), 2.050 (s, 12H), 1.995 (s, 12H), 1.062 (m, 20H). 13C NMR (CDCl3): δ 170.63, 170.07, 169.73, 145.06, 144.99, 144.86, 144.84, 144.54, 124.19, 124.14, 97.65, 69.99, 69.95, 69.56, 69.52, 69.48, 69.44, 69.03, 68.48, 67.23, 67.18, 66.05, 62.41, 50.16, 50.13, 50.01, 20.92, 20.77, 20.74, 20.71. HRMS calculated for [C95H141N13O50S]+, 1148.4315; found, 1148.4408. Compound 11. D-Biotin (7.6 mg, 0.031 mmol) was dissolved in N,N-dimethylformamide (DMF; 1 mL) and cooled to 0 °C. CDMT (6.0 mg, 0.03 mmol) and NMM (5 µL, 0.04 mmol) were added and stirred overnight at 0 °C. 10 (36 mg, 0.02 mmol) was dissolved in THF (1 mL) and added to the activated biotin. The mixture was stirred for 48 h, slowly warming to RT. Upon completion (TLC), the solvent was evaporated, and the residue was dissolved in EtOAc and washed with saturated NaHCO3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo, and the product was purified by flash column chromatography, eluting with a 90:10 mixture of CH2Cl2 and MeOH to give 11 as a light yellow syrupy solid (50 mg, 75%). 1H NMR (CDCl3): δ 7.90 (s, 1H), 7.84 (s, 1H), 7.81 (s, 1H), 7.76 (s, 1H), 6.02 (bs, 1H), 5.50 (bs, 1H), 5.31-5.24 (m, 13H), 5.00-4.70 (m, 12H), 4.65-4.51(m, 12H), 4.30-4.27 (m, 5H), 3.91 (m, 10H), 3.75 (m, 10H), 3.66-3.64 (m, 12H), 3.52 (m, 3H), 3.42 (m, 2H), 3.20 (m, 1H), 2.91 (m, 2H), 2.60 (m, 3H), 2.5 (bs, 3H), 2.33 (s, 3H), 2.15 (s, 12H), 2.09 (s, 12H), 2.05 (s, 12H), 1.99 (s, 12H), 1.68-1.48 (m, 15H). 13C NMR

(19) Wolfenden, M. L.; Cloninger, M. J. J. Am. Chem. Soc. 2005, 127, 12168– 12169. (20) Lee, R. T.; Lee, Y. C. Carbohydr. Res. 1974, 37, 193–201.

(21) Cheng, H.; Cao, X.; Xian, M.; Fang, L.; Cai, T. B.; Ji, J. J.; Tunac, J. B.; Sun, D.; Wang, P. G. J. Med. Chem. 2005, 48, 645–652.

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(CDCl3): 173.23, 170.66, 170.09, 169.98, 169.75, 168.41, 163.84, 159.59, 145.13, 145.00, 144.47, 124.22, 124.14, 124.04, 97.70, 97.43, 74.79, 69.99, 69.56, 69.47, 69.14, 69.05, 68.52, 67.23, 66.58, 66.33, 66.095, 65.81, 64.51, 64.41, 64.27, 62.44, 61.87, 60.191, 55.95, 55.38, 55.15, 53.45, 50.16, 50.09, 46.11, 40.58, 38.70, 35.72, 33.69, 31.93, 31.70, 30.16, 30.04, 29.71, 29.36, 29.32, 28.40, 28.02, 26.71, 25.41, 22.70, 20.936, 20.75, 14.14. HRMS calculated for [C115H155N13O51S]+, 1261.4703; found, 1261.4248. Compound GC1. 11 (30 mg, 0.012 mmol) was dissolved in MeOH (2 mL), and a solution of NaOMe in MeOH (0.5M, 0.5 mL) was added; the reaction mixture was stirred at RT for 6 h. The reaction was neutralized by careful addition of Amberlite-15 H+ resin, and the resin was filtered. The solvent was removed in vacuo, and the residue was purified by Biogel P-2 gel column chromatography, eluting with water, to give GC3 as a white solid (15 mg, 70%). 1H NMR (D2O): 8.44 (s, 1H), 8.11-8.06 (m, H), 4.95 (d, 1H), 4.75-4.54 (m, 13H), 4.40-4.36 (m, 1H), 4.11 (bs, 1H), 3.97-3.44 (m, 64H), 3.36-3.21 (m, 5H), 2.96-2.91 (m, 2H), 2.74-2.57 (m, 5H), 2.25-2.22 (m, 3H), 1.92-1.80 (m, 6H), 1.71-1.48 (m, 7H), 1.41-1.01 (m, 15H), 0.87 (bs, 5H). 13C NMR (D2O/CD3OD): 124.84, 100.21, 73.26, 71.16, 70.66, 69.76, 68.93, 67.24, 66.18, 63.70, 61.54, 50.05, 48.23, 48.03, 47.82, 47.61, 47.39, 47.19, 46.98, 46.43, 4.83. HRMS calculated for [C83H123N13O35S]+, 924.8843; found, 924.8937. Characterization of the Carbohydrate Coated Micrometer Magnetic Beads. The number of carbohydrates was determined by an anthrone-sulfuric acid assay as previously described by Lin et al.22 Briefly, carbohydrates (1-9 µg) were dissolved in 0.5 mL of deionized water. Next, 1 mL of a freshly prepared solution of 0.5% anthrone (w/w) in 95% sulfuric acid was added to 0.5 mL of ice cold sugar solution. The resulting solution was gently mixed and heated to 100 °C for 10 min and the allowed to cool to RT. The absorbance was recorded at 620 nm, and the plot of absorbance versus concentration of ligand resulted in a standard curve (Figure 1, Supporting Information). Next, different concentrations of the beads (0.5-2 mg) were subjected to the same method. From this concentration, the concentration of carbohydrates in 1 mg of beads was obtained and the number of carbohydrates per bead was calculated (Table 1, Supporting Information). Transverse Relaxation Measurements. All measurements were performed on a 1.41 T relaxometer (Minispec mq60, Bruker Optics Inc., The Woodlands, TX) according to manufacturer’s protocols. The relaxivity values of the carbohydrate coated magnetic beads were determined as described previously. The values are given in Table 1 (Supporting Information). The T2 measurements were performed at 40 °C. The experiments to obtain T2 for ConA are described (Figure 3A). Briefly, 1 mg of streptavidin coated micrometer magnetic beads (1 µm streptavidin coated Dynabeads MyOne, Invitrogen, Carlsbad, CA), which has the capacity to bind to ∼2500 pmol of free biotin, was washed three times with phosphate buffered saline (PBS) buffer (pH 7.4). Next, beads were incubated with 50 µg (0.10 µmol, 40-fold excess) of biotinylated R-mannoside, GC2 for 1 h at RT, followed by extensive washing and blocking with 0.1% BSA in PBS. For T2 measurements, 10 µg of the carbohydrate coated micrometer (22) Chien, Y. Y.; Jan, M. D.; Adak, A. K.; Tzeng, H. C.; Lin, Y. P.; Chen, Y. J.; Wang, K. T.; Chen, C. T.; Chen, C. C.; Lin, C. C. ChemBioChem 2008, 9, 1100–1109.

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beads was incubated with ConA for 1 h at RT, incubated at 40 °C for 10 min, and inserted into the relaxometer. In order to avoid increase in T2 induced by settling of magnetic beads, the beads were incubated with continuous stirring in the presence or absence of ConA. The ∆T2 was calculated by subtracting the T2 value in the presence of ConA from the T2 value in the absence of ConA at the indicated time intervals. The “Carr-Purcell-Meiboom-Gill” (CPMG) method was used for all the T2 measurements. The signals were acquired from 2 to 400 ms for the exponential decay profile from which T2 was calculated. The parameters used for the instrument are given in Supporting Information. Comparison of Different Concentrations of RCA120 in Presence and Absence of Antibody. To assess the ability of antibody to increase sensitivity, the T2 values were obtained for different concentrations of RCA120 lectin in the presence and absence of antibody specific to RCA120 lectin using biotinylated β-galactoside (GC3) coated micrometer magnetic beads (Figure 3). A graph of %∆T2 (% of maximum ∆T2) was plotted against RCA120 concentrations and fitted into a four parameter equation to obtain the EC50 value, Hill coefficient, and projected sensitivity concentration (minimum concentration detectable by the instrument) as described previously.23 The projected sensitivity concentration for GC3 was calculated to be 27 pM, which decreased to 4 pM using a combination of GC3 and antibody. RESULTS AND DISCUSSION To develop this bioassay, we synthesized tailored carbohydrates as the recognition element and coupled these molecules to commercial micrometer magnetic beads. Binding to multiple epitopes present on the target analyte promotes aggregation of the beads, resulting in measurable changes in the transverse relaxation time (T2) of surrounding water protons (Figure 1A). These changes can be readily monitored using a tabletop MRI scanner.24 Since the detection does not rely on an optical readout, complex human samples such as blood, urine, sputum, and environmental samples such as dirty water, mud, sewage, etc. can be analyzed with minimal sample preparation and no washing steps.24,25 Our strategy to observe aggregation was to measure changes in the transverse relaxation time (T2) of the water molecules in the solution containing the cognate protein-multivalent carbohydrate aggregates, since aggregated micrometer magnetic beads exhibit increased T2 values compared to nonaggregated micrometer beads. The relaxation time is dependent on the magnetic field induced aggregation and aggregation due to analyte-receptor binding.23 By measuring the T2 relaxation times over a period of time with and without analyte, we can observe receptor mediated aggregation events (Figure 1B). Interestingly, the size and iron content present in the magnetic probes affects the transverse relaxation times differently.23,26 Pioneering work by Weissleder and co-workers have shown that aggregated magnetic nanoparticles exhibited decreased T2 com(23) Koh, I.; Hong, R.; Weissleder, R.; Josephson, L. Angew. Chem., Int. Ed. Engl. 2008, 47, 4119–21. (24) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.; Weissleder, R. Nat. Biotechnol. 2002, 20, 816–820. (25) Kaittanis, C.; Naser, S. A.; Perez, J. M. Nano Lett. 2007, 7, 380–383. (26) Koh, I.; Hong, R.; Weissleder, R.; Josephson, L. Anal. Chem. 2009, 81, 3618–3622.

Scheme 1. Synthesis of the Biotinylated Tetra-Antennary Carbohydratea

Figure 1. (A) Schematic of the aggregation process caused by the interaction between multivalent carbohydrates and protein. The brown circles and red polygons represent micrometer beads with carbohydrate receptors and proteins, respectively. (B) The transverse relaxation (T2) values of micrometer magnetic beads (Inset: Difference in T2 in presence and absence of analyte is shown as ∆T2: red squares, in presence of specific analyte; blue triangles, in presence of nonspecific analyte). The magnetic beads were incubated with continuous stirring in the presence and absence of analyte to minimize settling. ∆T2 was calculated as a difference in T2 values in the presence and absence of analyte. a Reagents and conditions. (a) NaOMe, MeOH, RT, 12 h, 89%; (b) propargyl bromide, NaH, THF, reflux, 1 h, 51%; (c) sodium ascorbate, CuSO4, THF, H2O, RT, 48 h; (d) TIPS, TFA, CH2Cl2, RT, 24 h, 50% over two steps; (e) D-biotin, NMM, CDMT, THF, DMF, 75%; (f) NaOMe, MeOH, 70%.

Figure 2. Structures of the biotinylated molecules used in this study.

pared to dispersed magnetic nanoparticles, whereas aggregated magnetic microparticles exhibit increased T2 values. While the physical nature of this differential behavior on the T2 values is still under investigation, it is clear that the use of micrometer beads improves the limit of detection significantly.23 Therefore, we used micrometer beads in this study. For our initial studies, we synthesized carbohydrates that are known to bind to well characterized lectins. We synthesized R-mannosides, which bind to Concanavalin A (ConA) and Galanthus Nivalis lectin (GNL), and β-galactoside, which bind to Ricinus communis lectin (RCA120) and the plant toxin, ricin. Our previously described modular chemistry approach was used to construct the molecules depicted in Figure 2.3,5,17 The synthesis of one of the molecules, a tetra antennary carbohydrate bearing four mannose residues, GC1, is shown in Scheme 1. First, the acetate groups in known tetra-acetate β-galactoside 5 were deprotected under Zemple´n conditions to give

the deprotected product 6. Next, the hydroxyl groups were reacted with propargyl bromide in the presence of NaH to afford compound 7, which has four alkyne functionalities, in reasonable yield. The 1,3 dipolar cycloaddition of four equivalents of the known carbohydrate having azide functionality, 8 with 7 in the presence of sodium ascorbate and CuSO4, followed by deprotection of Boc group afforded compound 10 in 50% yield over two steps. The final steps included coupling of D-biotin to the free amine of 10 using standard coupling conditions followed by deprotection of acetate protecting groups to afford GC1 in appreciable yield. Next, we attached the ligands to streptavidin coated micrometer beads (1.0 µm streptavidin coated Dynabeads MyOne, Invitrogen, Carlsbad, CA). Incubation of the micrometer beads with excess biotinylated carbohydrates was followed by extensive washing and blocking with 0.1% BSA in PBS to reduce nonspecific binding. Next, we measured the relaxation properties of the carbohydrate coated magnetic beads to ensure that the integral properties did not change because of the surface coating of organic material. We found that the magnetic properties of the carbohydrate encapsulated magnetic beads correlated very well to the beads without any carbohydrate coating (Table 1, Supporting Information), indicating that carbohydrate coating does not perturb the magnetic properties of the beads. The relaxivity values of the magnetic beads were determined using the Bruker minispec 60 MHz mq60 (1.41 T) table top contrast agent analyzer as described previously.23,27 We also quantified the number of carbohydrates present on the bead using a chemically destructive method (Figure 1, Supporting Information). We found that the number of carbohydrates on the surface correlates (27) Hong, R.; Cima, M. J.; Weissleder, R.; Josephson, L. Magn. Reson. Med. 2008, 59, 515–520.

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Table 1. Specific Antibody to RCA120 Improves Sensitivity of GC3 (β-Galactoside Coated Beads)

without antibody with antibody

Figure 3. (A) Differentiation of R-mannoside binding proteins using GC2 coated micrometer magnetic beads: 1.9 nM GNL, 1.9 nM ConA, and 1.7 nM RCA120 were used. (B) Differentiation of β-galactoside binding proteins using GC3 coated micrometer magnetic beads: 1.7 nM ricin, 1.7 nM RCA120, and 1.9 nM ConA were used. (C) Increase in the sensitivity of the detection by increasing receptor density. Tetrameric GC1 and monomeric GC2 coated beads were incubated with 1.9 nM ConA. (D) Increase in sensitivity and selectivity system using a combination of carbohydrate and antibody. Orange squares: GC3 coated beads with 0.85 nM RCA120 and 100 µL of antibody to RCA120 (2 µg in 5 mL of PBS buffer); green triangles: 0.85 nM RCA120; purple triangles: 0.85 nM RCA120 and 100 µL of antibody to Stx1 (2 µg in 5 mL PBS of buffer). ∆T2 represents the difference in T2 values in between a control tube (no analyte) and an experimental tube. Results are the average of three independent trials. A linear increase in ∆T2 was observed for first 55 min. This was used to compute the initial ∆T2/min. (Please see Table 2, Supporting Information.)

very well with the manufacturer’s specifications. However, coating with the tetra-antennary R-mannoside, GC1, was considerably less compared to the monoantennary R-mannoside GC2, presumably because of the larger size of the former. Next, we tested the hypothesis that carbohydrate induced aggregation could lead to measurable changes in T2 values. Addition of ConA to GC2 (R-mannoside coated beads) resulted in the expected increase in T2 (filled blue circles, Figure 3). All the data is represented as ∆T2, which represents the difference in T2 values in between a control tube with no analyte and an experimental tube with the analyte incubated under identical conditions. We found no increase in ∆T2 when RCA120, the lectin that does not bind to R-mannosides, was used, indicating specificity (filled green triangles, Figure 3A). GNL, a lectin that is also known to bind to R-mannosides, resulted in a different initial aggregation rates with GC2 (filled red squares, Figure 3A). Statistical analysis of the data at 85 min revealed that these differences are statistically significant as measured by the students t test. (Figure 4A Supporting Information) These different aggregation rates are presumably due to differences in the affinity, number, and spatial arrangement of the binding sites in ConA and GNL (Figure 2, Supporting Information). At pH 7, ConA exists as a tetramer with four binding sites arranged at the apex of dome 7434

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EC50 [pM]

Hill slope

PSC [pM]

620 120

1.00 0.91

27 4

shaped monomers,28 whereas the structure of GNL tetramer resembles a flattened crown with 12 mannose binding sites.29,30 The binding of GC3 (β-galactoside coated beads) with RCA120 lectin is shown in Figure 3B (filled green triangles). As expected, there is a significant increase in the ∆T2 values, whereas the control analyte, ConA, exhibited no binding (filled blue triangles, Figure 3B). The initial aggregation rate of ricin, a protein that also recognizes β-galactosides, is depicted by the black filled squares (Figure 3B). As observed previously with the ConA and GNL system, RCA120 and ricin, two proteins that bind to the same carbohydrate, display different initial aggregation rates, which are statistically significant (Figure 4B, Supporting Information). We also studied the effect of increasing the number of carbohydrate receptors on the magnetic beads. ∆T2 values for GC1, which is the carbohydrate with four β mannosides (pink line, Figure 3C) are larger than monomeric R-mannoside GC2 (blue line, Figure 3C). Presumably, the increase in the number of R-mannosides in GC1 leads to more rapid and larger aggregates. Next, we hypothesized that the use of an additional recognition molecule that recognized different epitopes of the protein, for example antibody, would lead to increased aggregation through increased cross-linking, which could lead to increased sensitivity. We also expected an increase in specificity since the two recognition elements bind to two different epitopes. To test this hypothesis, we incubated GC3 (β-galactoside coated beads) coated beads with RCA120 for 1 h, followed by addition of antibody to RCA120 or nonspecific antibody to Shiga toxin (Stx) and incubation for 1 h. As shown in Figure 3D, antibody to RCA120 promoted a significant increase in the ∆T2 values, while polyclonal antibody specific to Stx did not. Quantitative analysis using different concentrations of RCA120 lectin was performed to obtain the limit of detection. A graph of %∆T2 was plotted against RCA120 concentrations and fitted into a four parameter equation to obtain EC50 value, Hill coefficient, and projected sensitivity concentration (PSC) (Figure 5).23 The results are summarized in Table 1. When GC3 (β-galactoside coated beads) coated beads were used with only RCA120, the EC50 value was 0.62 nM, but when antibody was used, the EC50 value decreased to 0.12 nM. Thus, using a combination of recognition molecules, the sensitivity was increased 5-fold. To demonstrate the versatility and scope of this assay, we used our previously identified ligands to detect and differentiate between Shiga toxin (Stx1 and 2) isoforms. The five identical subunits of Stx form a pentamer with superficial pentaradial symmetry. Each subunit has three binding sites, so there are total of 15 potential binding sites per toxin. Stx1 binds to Pk trisaccharides and Stx2 prefers the (28) Bouckaert, J.; Hamelryck, T. W.; Wyns, L.; Loris, R. J. Biol. Chem. 1999, 274, 29188–29195. (29) Hester, G.; Kaku, H.; Goldstein, I. J.; Wright, C. S. Nat. Struct. Biol. 1995, 2, 472–479. (30) Barre, A.; Bourne, Y.; Van Damme, E. J.; Peumans, W. J.; Rouge, P. Biochimie 2001, 83, 645–651.

limit of commercial enzyme linked immunosorbent assay (ELISA) kits, which uses several reagents and steps, to be 1 ng/mL. The magnetic relaxation switch method represents a 1000 fold increase in sensitivity over ELISA and, additionally, is a one step, no wash, add-and-measure technique.

Figure 4. (A,B) Selective detection of Stx variants using Pk and NHAc-Pk coated micrometer magnetic beads. Ten micrograms of beads and 100 ng of Stx were used. (C) Detection of samples spiked with Stx2 using NHAc-Pk coated magnetic beads. 100 ng of Stx2 was used. (D) Detection of different concentrations of spiked Stx2 in stool samples. The data in C and D represents ∆T2 values at 115 min. Results are the average of three independent trials.

Figure 5. Results of %∆ T2 as a function of concentration of RCA120 in the presence (red squares) and absence of antibody (blue circle). The results are the average of three independent trials.

N-acetylated analogues (Figure 2, Pk and NHAc-Pk).5 For initial studies, unpurified toxin from bacterial cultures was used to test if the detection system works in complex mixtures. As previously observed, Stx1 bound specifically to Pk coated beads, whereas Stx2 did not bind at all (Figure 4A). In contrast, Stx2 bound to N-acetylated NHAc-Pk coated beads, whereas Stx1 did not bind at all (Figure 4B and Figure 4E,F of Supporting Information). Thus, we were able to detect and differentiate between these closely related toxins directly from crude bacterial cultures. Next, we spiked Stx2 in a variety of samples that include milk, grape juice, hamburger, and stool samples. As shown in Figure 4C, this technique can be used to detect toxins from a variety of samples with minimal interference. Finally, we performed a dose dependent study of Stx2 in spiked stool samples (Figure 4D). A slight decrease in ∆T2 values was observed at high toxin concentrations due to the decreased cross-linking that occurs when toxin is in excess of ligand. However, this should not be a problem for detection applications since diluting the unknown will bring the system back to ligand excess. At lower concentrations, we could detect 1 pg/mL Shiga toxin in spiked stool samples. In comparison, we previously found the detection

CONCLUSION In summary, we have developed a simple, efficient, and selective carbohydrate based magnetic relaxation switch assay for the detection and differentiation of carbohydrate binding proteins. Differences in the structure, number of binding sites, and topological arrangement of the binding sites in proteins lead to a different initial aggregation rate with the carbohydrate coated beads, which can potentially be exploited for rapid detection and differentiation of carbohydrate binding proteins. It is important to note that differentiation of lectins that bind to the same carbohydrate is only possible when the same molar concentrations of proteins are used. This problem can be overcome using two different recognition elements that bind to different epitopes on the lectin. Indeed, we have demonstrated that selectivity and sensitivity can be enhanced using carbohydrates and antibodies. Alternatively, tailored carbohydrates to bind specifically to a protein or a multiplex platform, where an array of carbohydrate ligands bind with varying degrees of affinity to a particular analyte, leading to a fingerprint “pattern of recognition” response for the analyte,31,32 can be used to differentiate between lectins that prefer the same monosaccharide. Finally, we demonstrated that magnetic relaxation switch assays can be used to detect toxins in a complex medium and out-perform ELISA assays. This assay can be adapted for use in a wide variety of biological processes that involve carbohydrate recognition, including normal cellular processes (development, immune regulation) and abnormal cellular processes (infectious diseases, cancer). ACKNOWLEDGMENT Financial support for this work was provided by NIAID (U01AI075498 A.A.W. and S.S.I. and R01-064893 to A.A.W.) and NSF (Career CHE-0845005, S.S.I.). S.S.I. thanks Professor William R. Heinemann and IDCAST (Institute for Development and Commercialization of Advanced Sensor Technology, Dayton, OH 45402) for use of the table top MRI instrument. NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on August 4, 2010, with an omission of a grant in the Acknowledgment. The corrected version was reposted on August 9, 2010. SUPPORTING INFORMATION AVAILABLE Details of the synthesis of carbohydrates, additional characterization data, and the magnetic relaxation assay. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 14, 2010. Accepted July 25, 2010. AC101579M (31) Wright, A. T.; Griffin, M. J.; Zhong, Z.; McCleskey, S. C.; Anslyn, E. V.; McDevitt, J. T. Angew. Chem., Int. Ed. Engl. 2005, 44, 6375–6378. (32) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318– 323.

Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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